CN103751211A - Methods and pharmaceutical compositions for healing wounds - Google Patents

Abstract

A pharmaceutical composition for inducing or accelerating a healing process of damaged skin or skin wounds, comprising, as an active ingredient, a therapeutically effective amount of insulin and at least one other agent acting in synergy with the insulin, and a pharmaceutically acceptable carrier suitable for topical administration of the pharmaceutical composition. A method for inducing or accelerating a healing process of a damaged skin or skin wound, the method comprising administering to the damaged skin or skin wound a therapeutically effective amount of insulin and at least one other agent that acts synergistically with the insulin to induce or accelerate the healing process of the damaged skin or skin wound.

Description

Translated fromChinese

用于愈合创伤的方法和药物组合物Methods and pharmaceutical compositions for healing wounds

本申请是申请号为“200910217138.6”，发明名称为“用于愈合创伤的方法和药物组合物”的发明专利申请的分案申请。This application is a divisional application of the invention patent application with the application number "200910217138.6" and the invention title "Method and Pharmaceutical Composition for Healing Wounds".

发明领域和背景Field and Background of the Invention

本发明涉及一种用于诱导和／或加速细胞增殖和／或细胞分化并因此而加速创伤(wounds)的愈合过程的方法和药物组合物。更具体而言，本发明涉及经调节的丝氨酸／苏氨酸蛋白激酶，也称为PKC的表达和／或活化，例如由膜运输和活化引起的，用于诱导和／或加速细胞增殖和／或细胞分化和／或细胞迁移从而加速创伤的愈合过程的用途。根据本发明的教导，这种表达的调节可以通过下述作用实现：(i)用PKC表达构建体转化创伤细胞；(ii)用顺式作用元件转化创伤细胞，该元件将被插入到创伤细胞内源PKC基因上游邻近位置；(iii)给予胰岛素以诱导创伤细胞中PKC的表达和／或活化；(iv)用胰岛素表达构建体转化创伤细胞，其产生的表达的和分泌的胰岛素可作为PKC的表达和／或活化的上调剂；(v)用顺式作用元件转化创伤细胞，该元件将被插入到创伤细胞内源胰岛素基因上游邻近位置，被表达和分泌的胰岛素可作为PKC的表达和／或活化的上调剂；(vi)向创伤中植入胰岛素分泌细胞；(vii)用反式作用因子，例如PDX1转化创伤细胞，以诱导内源胰岛素产生和分泌，所述胰岛素可作为PKC的表达和／或活化的上调剂；以及(viii)给创伤施用PKC调节剂。The present invention relates to a method and a pharmaceutical composition for inducing and/or accelerating cell proliferation and/or cell differentiation and thus accelerating the healing process of wounds. More specifically, the present invention relates to the expression and/or activation of regulated serine/threonine protein kinases, also known as PKCs, e.g. by membrane trafficking and activation, for inducing and/or accelerating cell proliferation and/or Or the use of cell differentiation and/or cell migration to accelerate the healing process of wounds. According to the teachings of the present invention, the regulation of this expression can be achieved by: (i) transforming wounding cells with a PKC expression construct; (ii) transforming wounding cells with a cis-acting element, which will be inserted into the wounding cells Adjacent positions upstream of the endogenous PKC gene; (iii) administration of insulin to induce expression and/or activation of PKC in wounded cells; (iv) transformation of wounded cells with an insulin expression construct that produces expressed and secreted insulin that can serve as PKC (v) transform wounded cells with a cis-acting element, which will be inserted into the wounded cell's endogenous insulin gene upstream and adjacent, and the expressed and secreted insulin can serve as the expression and and/or activated upregulators; (vi) implantation of insulin-secreting cells into the wound; (vii) transformation of wounded cells with trans-acting factors, such as PDX1, to induce endogenous insulin production and secretion, which acts as a PKC an upregulator of expression and/or activation; and (viii) administering a PKC modulator to the wound.

本发明正如任何上述方法所实现的那样，也可离体实施，以产生皮肤移植物。The present invention, as implemented by any of the methods described above, can also be practiced ex vivo to produce skin grafts.

创伤治疗最主要的目的是使创伤闭合。张开的皮肤创伤代表了一类主要的创伤，包括烧伤，神经性溃疡，褥疮性溃疡，静脉淤滞性溃疡，和糖尿病性溃疡。The main purpose of trauma therapy is to close the wound. Open skin wounds represent a major class of wounds, including burns, neuropathic ulcers, decubitus ulcers, venous stasis ulcers, and diabetic ulcers.

张开的皮肤创伤通常经过这样的过程愈合，包括六个主要要素：(i)发炎；(ii)成纤维细胞增殖；(iii)血管增殖；(iv)结缔组织合成；(v)形成上皮；和(vi)创伤收缩。当这些要素，无论是单独的还是作为整体，不能正确发挥作用的时候，则破坏了创伤的愈合。有多种因素都可以影响创伤愈合，包括营养不良，感染，药理学试剂(例如放线菌素和类固醇)，高龄和糖尿病[参见Hunt and Goodson，Current Surgical Diagnosis&Treatment(Way；Appleton&Lange)，pp.86-98(1988)]。Open skin wounds usually heal through a process that involves six major elements: (i) inflammation; (ii) fibroblast proliferation; (iii) vascular proliferation; (iv) connective tissue synthesis; (v) epithelialization; and (vi) trauma contraction. When these elements, either individually or as a whole, do not function properly, wound healing is disrupted. A variety of factors can affect wound healing, including malnutrition, infection, pharmacological agents (eg, actinomycin and steroids), advanced age, and diabetes [see Hunt and Goodson, Current Surgical Diagnosis & Treatment (Way; Appleton & Lange), pp. 86 -98 (1988)].

就糖尿病而言，糖尿病的特征就在于胰岛素信号传导(signaling)受到损伤，血浆葡萄糖升高，以及在几种特定组织上发生慢性的并发症的易感性。在所有糖尿病的慢性并发症中，导致足部溃烂的受损的创伤愈合是研究最少的。然而糖尿病患者的皮肤溃疡花费了大量人力和财力(29，30)。而且，足部溃烂以及而后的下肢截断是最常见的糖尿病患者住院的原因(30-33)。在糖尿病中，创伤愈合过程受到破坏，愈合的创伤的特征在于伤口强度减小。组织修复的缺陷与几种因素有关，包括神经病，血管病和感染。但是，尚不明了其它机制，通过该机制，与胰岛素信号传导异常相关的糖尿病状态破坏创伤的愈合并改变皮肤生理。In the case of diabetes, diabetes is characterized by impaired insulin signaling, elevated plasma glucose, and susceptibility to chronic complications in several specific tissues. Of all the chronic complications of diabetes, impaired wound healing leading to foot ulcers is the least studied. However, skin ulceration in diabetic patients costs a lot of human and financial resources (29, 30). Furthermore, foot ulceration followed by lower extremity amputation is the most common reason for hospitalization in diabetic patients (30-33). In diabetes, the wound healing process is disrupted and healed wounds are characterized by reduced wound strength. Deficits in tissue repair are associated with several factors, including neuropathy, vascular disease, and infection. However, other mechanisms by which the diabetic state associated with abnormalities in insulin signaling impairs wound healing and alters skin physiology are not yet understood.

还有一个普遍存在的问题是身体的各个部分的外科手术之后的创伤愈合，手术成功但是创伤切口不愈合。There is also a common problem with wound healing following surgical procedures on various parts of the body where the surgery is successful but the wound incisions do not heal.

皮肤是分层的鳞状的上皮，其中正在进行生长和分化的细胞被严格隔开。在生理状态，增殖只限于附着于基底膜上的基底细胞。分化是一种空间过程，其中基底细胞失去与基底膜的附着，停止DNA合成并经历一系列形态学的和生物化学的变化。最终的成熟步骤是产生角质层，其形成皮肤的保护屏障(1，2)。在基底细胞开始分化的时候观察到的最早的变化与基底细胞从基底膜分离和迁移的能力有关(3)。创伤的愈合过程也有类似的变化，其中细胞迁移到创伤部位，增殖能力也增加。这些过程是皮肤层的重建和诱导表皮层正确分化所必需的。The skin is a stratified squamous epithelium in which growing and differentiating cells are tightly separated. Under physiological conditions, proliferation is restricted to basal cells attached to the basement membrane. Differentiation is a spatial process in which basal cells lose attachment to the basement membrane, cease DNA synthesis and undergo a series of morphological and biochemical changes. The final maturation step is the creation of the stratum corneum, which forms the skin's protective barrier (1, 2). The earliest changes observed when basal cells begin to differentiate relate to the ability of basal cells to detach and migrate from the basement membrane (3). Similar changes are seen in the healing process of wounds, in which cells migrate to the wound site and their ability to proliferate increases. These processes are necessary for the remodeling of the skin layers and for the induction of proper differentiation of the epidermal layer.

小鼠和人角质化细胞培养系统的开发大大方便了对表皮细胞生长和分化调节机制的分析(2，4)。在体外，角质化细胞可以保持为具有高生长速度的基底增殖细胞。而且，遵循表皮在体内的成熟模式可以在体外诱导分化。早期事件包括半桥粒组分的丧失(3，5)和α6β4整联蛋白的选择性丧失以及细胞与基质蛋白的附着的选择性丧失。这表明整联蛋白表达的变化是角质化细胞分化中的早期事件。早期的半桥粒接触(hemidesmosomal contact)的丧失导致角质化细胞向基底层上迁移，并且与培养的角质化细胞中以及皮肤中角蛋白1(K1)的诱导有关(1，3，6)。向颗粒层表型的进一步分化与β1和β4整联蛋白的表达的下调，与所有基质蛋白的附着潜力的丧失有关，然后就是角质化包膜的形成和细胞死亡。分化细胞最终从培养皿上脱落成为成熟鳞屑(2，7)。这种体外分化过程是严格遵循体内表皮成熟模式进行的。The development of mouse and human keratinocyte culture systems has greatly facilitated the analysis of mechanisms regulating epidermal cell growth and differentiation (2, 4). In vitro, keratinocytes can be maintained as basal proliferating cells with a high growth rate. Furthermore, differentiation can be induced in vitro following the maturation pattern of the epidermis in vivo. Early events include loss of hemidesmosomal components (3, 5) and selective loss of α6β4 integrins as well as selective loss of cellular attachment to matrix proteins. This suggests that changes in integrin expression are an early event in keratinocyte differentiation. Loss of early hemidesmosomal contact leads to suprabasal migration of keratinocytes and is associated with induction of keratin 1 (K1 ) in cultured keratinocytes as well as in skin (1, 3, 6). Further differentiation to the granular layer phenotype was associated with downregulation of expression of β1 and β4 integrins, loss of attachment potential of all matrix proteins, followed by formation of the cornified envelope and cell death. Differentiated cells eventually detach from the dish as mature scales (2, 7). This in vitro differentiation process strictly followed the in vivo epidermal maturation model.

最近对于角质化细胞生物学的研究突出了蛋白激酶C(ProteinKinase C)途径的贡献，其调节皮肤增殖和分化。丝氨酸-苏氨酸激酶的蛋白激酶C(PKC)家族在多种生物现象中都起着重要的调节作用(8，9)。PKC家族由至少12个单独的同种型(isoform)构成，其属于3个不同的类别：(i)传统同种型(α，β1，β2，γ)，其被磷脂酶C在胞内释放的Ca²⁺，佛波酯和二酰甘油激活；(ii)新同种型(δ，ε，η，θ)，其也可以被佛波酯和二酰甘油激活，但不能被Ca²⁺激活；(iii)家族的非典型性(ζ，λ，ι)成员，其不能被Ca²⁺，佛波酯或二酰甘油激活。Recent studies of keratinocyte biology have highlighted the contribution of the Protein Kinase C pathway, which regulates skin proliferation and differentiation. The protein kinase C (PKC) family of serine-threonine kinases play important regulatory roles in a variety of biological phenomena (8,9). The PKC family consists of at least 12 individual isoforms (isoforms), which belong to 3 distinct classes: (i) traditional isoforms (α, β1, β2, γ), which are released intracellularly by phospholipase C Ca²⁺ , activated by phorbol esters and diacylglycerols; (ii) new isoforms (δ, ε, η, θ), which can also be activated by phorbol esters and diacylglycerols, but not by Ca²⁺ Activation; (iii) atypical (ζ,λ,ι) members of the family, which cannot be activated by Ca²⁺ , phorbol esters or diacylglycerols.

当被激活的时候，大部分但不是所有同种型被认为会从细胞质迁移到质膜上。同种型的类型和分布模式在不同的组织中各不相同，也会随表型而变化。由于PKC在多种激素作用的细胞终点中的重要性，已有多项研究阐明了其结构和功能。在体内和培养物中已识别了皮肤中的五种PKC同种型-α，δ，ε，η和ζ。近来的研究表明PKC信号传导途径是分化反应的主要胞内调节剂(10，11)。而且，PKC的药理学活化剂是体内和体外角质化细胞分化的强有力的诱导剂(4，12)，PKC抑制剂能防止分化标志物的表达(10)。When activated, most but not all isoforms are thought to migrate from the cytoplasm to the plasma membrane. The type and distribution pattern of isotypes vary across different tissues and also vary with phenotype. Because of the importance of PKC in the cellular endpoints of multiple hormone actions, several studies have elucidated its structure and function. Five PKC isoforms in skin - α, δ, ε, η and ζ - have been identified in vivo and in culture. Recent studies have shown that the PKC signaling pathway is a major intracellular regulator of differentiation responses (10, 11). Furthermore, pharmacological activators of PKC are potent inducers of keratinocyte differentiation in vivo and in vitro (4, 12), and PKC inhibitors prevent the expression of differentiation markers (10).

在构思本发明时，猜测PKC同种型的过表达和／或活化可能会有助于加速创伤愈合过程。由于难以有效地用传统方法向原代细胞中引入外源基因，因而对各个PKC同种型在皮肤细胞增殖和／或分化中的作用的研究受到了限制。生命期短，存在分化可能以及不能分离到稳定的转化体都使得外源基因不能有效转导入原代皮肤细胞。In conceiving the present invention, it was hypothesized that overexpression and/or activation of PKC isoforms might help to accelerate the wound healing process. Studies of the role of individual PKC isoforms in skin cell proliferation and/or differentiation have been limited by the difficulty of efficiently introducing foreign genes into primary cells using traditional methods. The short lifespan, the possibility of differentiation and the inability to isolate stable transformants all make it impossible to effectively transfer foreign genes into primary skin cells.

现有技术描述了胰岛素作为创伤愈合的治疗剂的潜在应用。美国专利5,591,709，5,461,030和5,145,679描述了将胰岛素局部施用于创伤以促进创伤愈合。但是，这些专利所描述的都是将胰岛素和葡萄糖组合使用，因为胰岛素的功能是增加葡萄糖的吸收，因而促进创伤愈合。The prior art describes the potential use of insulin as a therapeutic agent for wound healing. US Patents 5,591,709, 5,461,030 and 5,145,679 describe the topical application of insulin to wounds to promote wound healing. However, these patents all describe the combination of insulin and glucose, because the function of insulin is to increase the absorption of glucose, thereby promoting wound healing.

美国专利申请09／748,466和国际专利申请PCT／US98／21794描述了局部施用于皮肤以促进皮肤健康或治疗较浅的皮肤损伤的含有胰岛素的组合物。但是，这些专利申请并没有教导使用胰岛素治疗慢性的，II度(Grade II)或较深的创伤。US Patent Application 09/748,466 and International Patent Application PCT/US98/21794 describe compositions containing insulin that are topically applied to the skin to promote skin health or treat shallower skin lesions. However, these patent applications do not teach the use of insulin to treat chronic, Grade II or deeper wounds.

国际专利申请PCT／US01／10245描述了将氰基丙烯酸酯聚合物密封剂与胰岛素或银组合使用以使创伤愈合。但是，在此申请中既没有教导也没有暗示胰岛素和另一种生物活性试剂组合使用可以调节PKC的表达和／或活化。International Patent Application PCT/US01/10245 describes the use of cyanoacrylate polymer sealants in combination with insulin or silver to heal wounds. However, there is neither teaching nor suggestion in this application that the combination of insulin and another biologically active agent can modulate the expression and/or activation of PKC.

国际专利申请PCT／US85／00695描述了局部施用胰岛素以治疗糖尿病。但是，该专利申请并没有教导使用胰岛素治疗与糖尿病无关的创伤。International patent application PCT/US85/00695 describes the topical administration of insulin for the treatment of diabetes. However, this patent application does not teach the use of insulin to treat wounds not related to diabetes.

国际专利申请PCT／US92／03086描述了可以含有胰岛素的治疗用微乳制剂。但是，其公开的内容没有教导使用胰岛素制剂使创伤愈合。International Patent Application PCT/US92/03086 describes therapeutic microemulsion formulations which may contain insulin. However, its disclosure does not teach the use of insulin preparations for wound healing.

美国专利4,673,649和4,940,660描述了用于人角质化细胞和表皮细胞体外无性系生长的组合物，其中含有表皮生长因子和胰岛素。这些专利教导了将胰岛素用于培养的皮肤细胞的发育，所述细胞可用于移植。但是，这些专利并未教导将胰岛素在体内用于创伤。US Patents 4,673,649 and 4,940,660 describe compositions for the in vitro clonal growth of human keratinocytes and epidermal cells containing epidermal growth factor and insulin. These patents teach the use of insulin for the development of cultured skin cells that can be used for transplantation. However, these patents do not teach the use of insulin in vivo for trauma.

上述引用的现有技术参考文献并未教导或暗示将胰岛素用于调节PKC的表达和／或活化，从而加速创伤的愈合过程。而且，现有技术并未教导或暗示使用核酸构建体或基因转化技术向创伤提供胰岛素，从而加速创伤的愈合过程。The prior art references cited above do not teach or suggest the use of insulin to modulate the expression and/or activation of PKC to accelerate the wound healing process. Furthermore, the prior art does not teach or suggest the use of nucleic acid constructs or gene transformation techniques to deliver insulin to wounds to accelerate the wound healing process.

目前普遍意识到需要，并且为了便利之目的也需要能加速创伤愈合相关过程的新方法。此外，还普遍意识到需要，并且为了便利之目的也需要能将重组基因插入到皮肤细胞中的有效方法，其将加速细胞增殖和／或分化过程和创伤愈合。There is a general awareness of the need, and for convenience, of new methods that can speed up the processes associated with wound healing. In addition, there is a generally recognized need, and a need for convenience, for efficient methods of inserting recombinant genes into skin cells that will accelerate cell proliferation and/or differentiation processes and wound healing.

发明概述Summary of the invention

当实施本发明的时候，发明人发现将胰岛素单独施用到创伤处会产生不良的副作用例如过度血管生成，发炎，上皮细胞增生和疤痕形成(见下文实施例部分的实施例23)。本发明人进一步发现将胰岛素和一种或多种能在定居于创伤部位的细胞(cells colonizing thewound area)中调节PKC的表达和／或活性的试剂组合使用可有效避免胰岛素引起的副作用，并且同时显著加速创伤愈合过程。When practicing the present invention, the inventors found that administration of insulin alone to the wound produced undesirable side effects such as excessive angiogenesis, inflammation, epithelial cell proliferation and scarring (see Example 23 in the Examples section below). The present inventors further found that the combination of insulin and one or more agents that can regulate the expression and/or activity of PKC in cells colonizing the wound area can effectively avoid the side effects caused by insulin, and at the same time Significantly accelerates the wound healing process.

本发明提供了高效治疗创伤并且没有不良副作用的新方法和组合物，包括向创伤部位提供有效量的胰岛素和／或其它能在创伤部位的细胞中调节PKC的表达和／或活性并与胰岛素有协同作用的试剂以加速创伤愈合的过程。The present invention provides a new method and composition for effectively treating wounds without adverse side effects, including providing effective doses of insulin and/or other substances capable of regulating the expression and/or activity of PKC in the cells of the wound site and interacting with insulin to the wound site. Agents that act synergistically to accelerate the process of wound healing.

因此，根据本发明的一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括给损伤的皮肤或皮肤创伤施用治疗有效量的调节PKC产生和／或PKC活化的试剂的步骤。Therefore, according to one aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising administering to damaged skin or skin wounds a therapeutically effective amount of a PKC production-regulating and/or Procedure for reagents for PKC activation.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的至少一种调节PKC产生和／或活性的试剂；和药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of at least one an agent that modulates PKC production and/or activity; and a pharmaceutically acceptable carrier.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括给损伤的皮肤或皮肤创伤施用治疗有效量的胰岛素和至少一种与胰岛素有协同作用的其它试剂的步骤，以诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, the method comprising administering to the damaged skin or skin wounds a therapeutically effective amount of insulin and at least one A step in which other agents act synergistically to induce or accelerate the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的胰岛素，至少一种能与胰岛素协同作用的其它试剂，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of insulin, at least An additional agent capable of synergistically acting with insulin, and a pharmaceutically acceptable carrier designed for topical administration of said pharmaceutical composition.

根据本发明的另一个方面提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括给损伤的皮肤或皮肤创伤施用单次剂量的治疗有效量的胰岛素的步骤，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising the step of administering a single dose of a therapeutically effective amount of insulin to the damaged skin or skin wounds, Thereby inducing or accelerating the healing process of damaged skin or skin wounds.

根据本发明的另一个方面提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，所选择的能诱导或加速损伤的皮肤或皮肤创伤的愈合过程的单一剂量单位的胰岛素，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a selected substance capable of inducing or accelerating damage. A single dosage unit of insulin for the healing process of skin or skin wounds, and a pharmaceutically acceptable carrier designed for topical administration of said pharmaceutical composition.

根据本发明的另一个方面提供了一种诱导或加速陈旧皮肤创伤(old skin wound)的愈合过程的方法，所述方法包括给陈旧皮肤创伤施用单次剂量的治疗有效量的胰岛素的步骤，从而诱导或加速陈旧皮肤创伤的愈合过程。According to another aspect of the present invention there is provided a method of inducing or accelerating the healing process of an old skin wound, said method comprising the step of administering a single dose of a therapeutically effective amount of insulin to the old skin wound, whereby Induces or accelerates the healing process of old skin wounds.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括向损伤的皮肤或皮肤创伤植入治疗有效量的胰岛素分泌细胞的步骤，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising the step of implanting a therapeutically effective amount of insulin-secreting cells into the damaged skin or skin wounds, Thereby inducing or accelerating the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的胰岛素分泌细胞，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, the pharmaceutical composition comprising, as an active ingredient, insulin-secreting cells, and designed for A pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括转化损伤的皮肤或皮肤创伤的细胞以产生和分泌胰岛素的步骤，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising the step of transforming cells of damaged skin or skin wounds to produce and secrete insulin, thereby inducing Or to accelerate the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，设计用于转化损伤的皮肤或皮肤创伤处细胞以产生和分泌胰岛素的核酸构建体，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a drug designed to transform damaged skin Or a nucleic acid construct for producing and secreting insulin by cells at a skin wound, and a pharmaceutically acceptable carrier designed for local administration of the pharmaceutical composition.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括转化损伤的皮肤或皮肤创伤处细胞以产生蛋白激酶C的步骤，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method for inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising the step of transforming damaged skin or skin wound cells to produce protein kinase C, thereby inducing Or to accelerate the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，设计用于转化损伤的皮肤或皮肤创伤处细胞以产生蛋白激酶C的核酸构建体，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a drug designed to transform damaged skin or skin wound cells to produce a nucleic acid construct of protein kinase C, and a pharmaceutically acceptable carrier designed for local administration of the pharmaceutical composition.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括给损伤的皮肤或皮肤创伤施用治疗有效量的PKC活化剂的步骤，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method for inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising the step of applying a therapeutically effective amount of a PKC activator to damaged skin or skin wounds, thereby Induces or accelerates the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的PKC活化剂，以诱导或加速损伤的皮肤或皮肤创伤的愈合过程，以及药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of a PKC activator , to induce or accelerate the healing process of damaged skin or skin wounds, and a pharmaceutically acceptable carrier.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括给损伤的皮肤或皮肤创伤施用治疗有效量的共聚物-1的步骤。According to another aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, the method comprising the step of administering a therapeutically effective amount of Copolymer-1 to the damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的共聚物-1，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of a copolymer- 1, and a pharmaceutically acceptable carrier designed for topical administration of said pharmaceutical composition.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的共聚物-1，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of a copolymer- 1, and a pharmaceutically acceptable carrier designed for topical administration of said pharmaceutical composition.

根据本发明的另一个方面，提供了一种诱导或加速损伤的皮肤或皮肤创伤的愈合过程的方法，所述方法包括调节定居于损伤的皮肤或皮肤创伤处真皮细胞中的至少一种PKC同种型的表达和／或活性；给所述真皮细胞施用治疗有效量的至少一种选自激素，生长因子，脂肪细胞激素，PKCδRACK和GW9662的其它试剂以及调节所述PKC同种型的表达和／或活性，从而诱导或加速损伤的皮肤或皮肤创伤的愈合过程。According to another aspect of the present invention, there is provided a method of inducing or accelerating the healing process of damaged skin or skin wounds, said method comprising modulating at least one PKC in dermal cells residing at damaged skin or skin wounds and simultaneously isoform expression and/or activity; administering to said dermal cells a therapeutically effective amount of at least one other agent selected from hormones, growth factors, adipocyte hormones, PKCδRACK and GW9662 and modulating said PKC isoform expression and and/or active, thereby inducing or accelerating the healing process of damaged skin or skin wounds.

根据本发明的另一个方面，提供了一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的，治疗有效量的调节至少一种PKC同种型的表达或活性的物质，和至少一种选自激素，生长因子，脂肪细胞激素，PKCδRACK和GW9662的其它试剂，以及药学可接受的载体。According to another aspect of the present invention, there is provided a pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of modulating at least one A substance for the expression or activity of a PKC isoform, and at least one other agent selected from hormones, growth factors, adipocyte hormones, PKCδRACK and GW9662, and a pharmaceutically acceptable carrier.

根据下述的发明的优选实施方案的其它特征，所述创伤选自溃疡，糖尿病相关的创伤，烧伤，晒伤，老化皮肤创伤，角膜溃烂创伤，炎性胃肠道疾病创伤，肠炎性疾病创伤，克罗恩氏病创伤，溃疡性结肠炎，痔疮，大疱性表面松解症创伤，皮肤起疱创伤，牛皮癣创伤，saborehic皮炎创伤，动物皮肤创伤，动物糖尿病创伤，视网膜病创伤，口腔创伤(粘膜炎)，阴道粘膜炎创伤，牙龈病创伤，裂伤(laceration)，手术切口创伤和手术后粘连创伤(adhesis wound)。According to further features in preferred embodiments of the invention described below, the wound is selected from the group consisting of ulcers, diabetes-related wounds, burns, sunburns, aged skin wounds, corneal ulcerative wounds, inflammatory gastrointestinal disease wounds, intestinal inflammatory disease wounds , Crohn's disease wounds, ulcerative colitis, hemorrhoids, bullous surface lysis wounds, skin blistering wounds, psoriatic wounds, saborehic dermatitis wounds, animal skin wounds, animal diabetic wounds, retinopathy wounds, oral wounds (mucositis), vaginal mucositis wounds, gum disease wounds, lacerations, surgical incision wounds and postoperative adhesion wounds.

根据所述的优选实施方案的其它特征，所述溃疡是糖尿病性溃疡，褥疮，静脉性溃疡，胃溃疡和HIV相关溃疡。According to still further features in the described preferred embodiments the ulcer is a diabetic ulcer, a decubitus ulcer, a venous ulcer, a gastric ulcer and an HIV-associated ulcer.

根据所述的优选实施方案的其它特征，所述胰岛素是重组的。According to still further features in the described preferred embodiments the insulin is recombinant.

根据所述的优选实施方案的其它特征，所述胰岛素是天然来源的。According to still further features in the described preferred embodiments the insulin is of natural origin.

根据所述的优选实施方案的其它特征，所述其它试剂是血小板衍生的生长因子。According to still further features in the described preferred embodiments the other agent is platelet derived growth factor.

根据所述的优选实施方案的其它特征，所述其它试剂是PKC-α抑制剂。According to still further features in the described preferred embodiments the other agent is a PKC-alpha inhibitor.

根据所述的优选实施方案的其它特征，给药是单次施用。According to still further features in the described preferred embodiments the administering is a single administration.

根据所述的优选实施方案的其它特征，所述陈旧皮肤创伤是至少2天龄(2days old)的。According to still further features in the described preferred embodiments the old skin wound is at least 2 days old.

根据所述的优选实施方案的其它特征，所述胰岛素浓度为0.1μM到10μM。根据所述的优选实施方案的其它特征，胰岛素的剂量单位是在0.01-0.2ml的药物组合物中的0.001到5nM。According to still further features in the described preferred embodiments the insulin concentration is from 0.1 μM to 10 μM. According to still further features in the described preferred embodiments the dosage unit of insulin is 0.001 to 5 nM in 0.01-0.2 ml of the pharmaceutical composition.

根据所述的优选实施方案的其它特征，所述胰岛素的剂量为在0.01-0.2ml的药物组合物中的0.01到0.5nM。According to still further features in the described preferred embodiments the dose of insulin is 0.01 to 0.5 nM in 0.01-0.2 ml of the pharmaceutical composition.

根据所述的优选实施方案的其它特征，所述药物组合物选自水溶液，凝胶，乳剂，糊剂，洗剂，喷雾，悬液，粉末，分散剂，油膏剂和软膏剂。According to still further features in the described preferred embodiments the pharmaceutical composition is selected from the group consisting of aqueous solutions, gels, emulsions, pastes, lotions, sprays, suspensions, powders, dispersions, salves and ointments.

根据所述的优选实施方案的其它特征，所述药物组合物含有固体支持物。According to still further features in the described preferred embodiments the pharmaceutical composition comprises a solid support.

根据所述的优选实施方案的其它特征，所述细胞被转化以产生和分泌胰岛素。According to still further features in the described preferred embodiments the cells are transformed to produce and secrete insulin.

根据所述的优选实施方案的其它特征，所述细胞被重组PDX1基因转化，从而使得所述细胞产生和分泌天然的胰岛素。According to still further features in the described preferred embodiments the cells are transformed with the recombinant PDX1 gene such that the cells produce and secrete native insulin.

根据所述的优选实施方案的其它特征，所述细胞被顺式作用元件序列转化，该序列整合到所述细胞的内源胰岛素基因的上游，从而所述细胞产生和分泌天然胰岛素。According to still further features in the described preferred embodiments the cell is transformed with a cis-acting element sequence integrated upstream of the cell's endogenous insulin gene such that the cell produces and secretes native insulin.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞能够形成分泌颗粒。According to still further features in the described preferred embodiments the insulin secreting cells are capable of forming secretory granules.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞是内分泌细胞。According to still further features in the described preferred embodiments the insulin secreting cells are endocrine cells.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞是人源的。According to still further features in the described preferred embodiments the insulin secreting cells are of human origin.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞是组织相容性人化动物来源的。According to still further features in the described preferred embodiments the insulin secreting cells are of histocompatible humanized animal origin.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞分泌人胰岛素。According to still further features in the described preferred embodiments the insulin secreting cells secrete human insulin.

根据所述的优选实施方案的其它特征，所述胰岛素分泌细胞是自体细胞。According to still further features in the described preferred embodiments the insulin secreting cells are autologous cells.

根据所述的优选实施方案的其它特征，所述细胞选自成纤维细胞，上皮细胞和角质化细胞。According to still further features in the described preferred embodiments the cells are selected from the group consisting of fibroblasts, epithelial cells and keratinocytes.

根据所述的优选实施方案的其它特征，所述细胞被转化以产生蛋白激酶C转录激活剂，从而使得所述细胞产生天然的蛋白激酶C。According to still further features in the described preferred embodiments the cell is transformed to produce a protein kinase C transcriptional activator such that the cell produces native protein kinase C.

根据所述的优选实施方案的其它特征，所述细胞被顺式作用元件序列转化，该序列整合到所述细胞的内源蛋白激酶C的上游，从而使得所述细胞产生天然的蛋白激酶C。According to still further features in the described preferred embodiments the cell is transformed with a cis-acting element sequence integrated upstream of endogenous protein kinase C of the cell such that the cell produces native protein kinase C.

根据所述的优选实施方案的其它特征，所述细胞被重组蛋白激酶C基因转化，从而使得所述细胞产生重组蛋白激酶C。According to still further features in the described preferred embodiments the cell is transformed with a recombinant protein kinase C gene such that the cell produces recombinant protein kinase C.

根据所述的优选实施方案的其它特征，所述蛋白激酶C选自PKC-β1，PKC-β2，PKC-γ，PKC-θ，PKC-λ，和PKC-ι。According to still further features in the described preferred embodiments the protein kinase C is selected from the group consisting of PKC-β1, PKC-β2, PKC-γ, PKC-θ, PKC-λ, and PKC-ι.

根据所述的优选实施方案的其它特征，所述蛋白激酶C选自PKC-α，PKC-δ，PKC-ε，PKC-η和PKC-ζ。According to still further features in the described preferred embodiments the protein kinase C is selected from the group consisting of PKC-alpha, PKC-delta, PKC-epsilon, PKC-eta and PKC-zeta.

根据所述的优选实施方案的其它特征，在适于局部施用的药物组合物中含有共聚物-1。According to still further features in the described preferred embodiments Copolymer-1 is contained in a pharmaceutical composition suitable for topical administration.

根据所述的优选实施方案的其它特征，所述PKC同种型选自PKC-α，PKC-β，PKC-δ，和PKC-ζ。According to still further features in the described preferred embodiments the PKC isoform is selected from the group consisting of PKC-alpha, PKC-beta, PKC-delta, and PKC-zeta.

根据所述的优选实施方案的其它特征，所述激素是胰岛素。According to still further features in the described preferred embodiments the hormone is insulin.

根据所述的优选实施方案的其它特征，所述生长因子选自IL-6，KFG和TNFα。According to still further features in the described preferred embodiments the growth factor is selected from the group consisting of IL-6, KFG and TNF[alpha].

根据所述的优选实施方案的其它特征，所述脂肪细胞激素是adipsin或脂联素(adiponectin)。According to still further features in the described preferred embodiments the adipocyte hormone is adipsin or adiponectin.

本发明成功解决了现有的已知技术的缺点，提供了用于对抗损伤的皮肤或皮肤创伤的新的治疗方法。The present invention successfully overcomes the disadvantages of the prior known technology, providing a new treatment method for combating damaged skin or skin wounds.

附图简述Brief description of the drawings

本发明仅通过举例方式并参考所附的附图进行描述。现在具体参考附图的细节，应当指出所示出的细节仅仅是举例方式，用于对本发明的优选实施方案进行举例说明，而且是为了提供对本发明的原理和概念最有用的和最容易理解的描述。从这一点来说，除了对本发明有基本理解所必需的内容不会再对本发明进行更详细的结构性细节描述，对于附图的描述可使所属领域技术人员明了本发明的各种形式如何实施。The present invention has been described, by way of example only, with reference to the accompanying drawings. With specific reference now to the details of the drawings, it should be noted that those particulars shown are by way of example only, illustrative of preferred embodiments of the invention, and for the purpose of providing the most useful and understandable understanding of the principles and concepts of the invention. describe. In this regard, the invention will not be described in further structural detail except that which is necessary for a basic understanding of the invention, and the description of the accompanying drawings will enable those skilled in the art to understand how the various forms of the invention may be practiced. .

在附图中：In the attached picture:

图1证明了使用重组腺病毒载体可有效过表达PKC同种型：左边：四天龄的原代角质化细胞用β-gal腺病毒感染1小时。感染后48小时，固定细胞，通过诱导蓝色反应定量确定β-半乳糖苷酶蛋白的活化并与未感染的角质化细胞相比。右边：四天龄的原代角质化细胞用重组同种型特异性PKC腺病毒感染1小时。二十四小时后，提取感染培养物(Ad)和未感染对照培养物(C)的蛋白进行Western blot分析，用下述实施例部分所述的同种型特异性抗-PKC抗体分析样品。Figure 1 demonstrates efficient overexpression of PKC isoforms using recombinant adenoviral vectors: Left: Four-day-old primary keratinocytes were infected with β-gal adenovirus for 1 hour. Forty-eight hours after infection, the cells were fixed and the activation of the β-galactosidase protein was quantified by inducing a blue reaction and compared to uninfected keratinocytes. Right: Four-day-old primary keratinocytes were infected with recombinant isoform-specific PKC adenovirus for 1 hour. Twenty-four hours later, protein was extracted from infected cultures (Ad) and uninfected control cultures (C) for Western blot analysis, and samples were analyzed with isotype-specific anti-PKC antibodies as described in the Examples section below.

图2显示了苔藓虫素1(bryostatin1)对PKC的激活诱导过表达的PKC同种型的迁移。四天龄的原代角质化细胞用同种型特异性的重组PKC腺病毒感染1小时。感染后二十四小时，细胞不进行处理(C)或用苔藓虫素1(B)刺激30分钟，然后分级。将蛋白样品进行Westernblot，用同种型特异性抗-PKC抗体分析。Figure 2 shows that activation of PKC bybryostatin 1 induces migration of overexpressed PKC isoforms. Four-day-old primary keratinocytes were infected with isotype-specific recombinant PKC adenovirus for 1 hr. Twenty-four hours post-infection, cells were left untreated (C) or stimulated with bryostatin 1 (B) for 30 min and then graded. Protein samples were subjected to Western blot and analyzed with an isotype-specific anti-PKC antibody.

图3显示了过表达的PKC同种型的天然形式具有活性。四天龄的原代角质化细胞用同种型特异性重组PKC腺病毒感染1小时。感染后18个小时，将未感染的对照细胞(C)和PKC同种型过表达细胞(OE)的细胞裂解物用同种型特异性抗PKC抗体进行免疫沉淀反应。将免疫沉淀物用于进行PKC活性检测，如下述实施例部分所述。Figure 3 shows that native forms of overexpressed PKC isoforms are active. Four-day-old primary keratinocytes were infected with isotype-specific recombinant PKC adenovirus for 1 hr. Eighteen hours after infection, cell lysates of uninfected control cells (C) and PKC isoform overexpressing cells (OE) were immunoprecipitated with isotype-specific anti-PKC antibodies. Immunoprecipitates were used to perform PKC activity assays as described in the Examples section below.

图4证明了过表达的特异性PKC同种型诱导原代角质化细胞中的明显的形态学改变。原代角质化细胞不进行处理(C)或者用重组PKCα，δ，η，或ζ腺病毒感染。二十四小时后，用亮场显微镜对培养物进行观察并拍照(x20)。Figure 4 demonstrates that overexpression of specific PKC isoforms induces distinct morphological changes in primary keratinocytes. Primary keratinocytes were left untreated (C) or infected with recombinant PKCα, δ, η, or ζ adenovirus. Twenty-four hours later, the cultures were observed and photographed with a bright-field microscope (x20).

图5显示了过表达的PKC同种型在被感染的原代角质化细胞中的清楚定位。将原代角质化细胞接种在层粘连蛋白5(laminin5)涂覆的玻璃载玻片上。培养物不进行处理或者用不同的重组PKC腺病毒感染。感染后二十四小时，固定细胞，洗涤并风干。用同种型特异性抗PKC抗体然后用FITC结合的二抗对培养物进行免疫荧光分析。用共聚焦显微镜对细胞进行扫描，对典型视野进行拍照。Figure 5 shows the clear localization of overexpressed PKC isoforms in infected primary keratinocytes. Primary keratinocytes were seeded on laminin5-coated glass slides. Cultures were left untreated or infected with different recombinant PKC adenoviruses. Twenty-four hours after infection, cells were fixed, washed and air-dried. Cultures were subjected to immunofluorescence analysis with an isotype-specific anti-PKC antibody followed by a FITC-conjugated secondary antibody. Cells were scanned with a confocal microscope, and typical fields of view were photographed.

图6证明了PKC同种型特异性调节α6β4整联蛋白的表达。五天龄的原代小鼠皮肤角质化细胞不进行处理或者用PKCδ，PKCα，PKCη或PKCζ重组腺病毒感染。感染后四十八小时，用膜细胞组分进行SDS-PAGE电泳，转至硝酸纤维素滤膜上，用抗α6和抗β4抗体进行免疫印迹并用ECL进行分析。Figure 6 demonstrates that PKC isoform-specific regulation of α6β4 integrin expression. Five-day-old primary mouse skin keratinocytes were left untreated or infected with PKCδ, PKCα, PKCη or PKCζ recombinant adenoviruses. Forty-eight hours after infection, membrane cell fractions were subjected to SDS-PAGE electrophoresis, transferred to nitrocellulose filters, immunoblotted with anti-α6 and anti-β4 antibodies and analyzed by ECL.

图7显示了过表达的PKCη和PKCδ诱导角质化细胞的增殖。五天的原代小鼠皮肤角质化细胞不进行处理或者用PKCδ，PKCα，PKCη或PKCζ重组腺病毒感染。感染后四十八小时，进行1小时的³H-胸苷掺入以分析细胞增殖，如实验步骤中所述。结果表示为cpm／培养皿，与β-半乳糖苷酶感染的角质化细胞相比较。各个值表示为3次独立实验中三次重复测定值的平均值±标准差。Figure 7 shows that overexpressed PKCη and PKCδ induce proliferation of keratinocytes. Five day old primary mouse skin keratinocytes were left untreated or infected with PKCδ, PKCα, PKCη or PKCζ recombinant adenoviruses. Forty-eight hours post-infection, 1 hour^of3H -thymidine incorporation was performed to analyze cell proliferation as described in the experimental procedure. Results are expressed as cpm/dish and compared to β-galactosidase-infected keratinocytes. Individual values are expressed as the mean ± standard deviation of triplicate determinations in 3 independent experiments.

图8证明了PKC同种型的过表达对α6β4整联蛋白的半桥粒定位的作用。原代角质化细胞接种在层粘连蛋白5涂覆的玻璃载玻片上，将角质化细胞培养物在低Ca²⁺的MEM中保持48小时。然后培养物不进行处理(A)，或者被PKCα，PKCδ，PKCη或PKCζ重组腺病毒感染(分别是B-E)。感染后二十四小时，用4％多聚甲醛固定角质化细胞，然后用0.2％Triton-X-100温和提取，用PBS洗涤并风干，如实验步骤中所述。用同种型特异性抗α6抗体然后用FITC结合的二抗对培养物进行免疫荧光分析，如实验步骤中所述。Figure 8 demonstrates the effect of overexpression of PKC isoforms on hemidesmosomal localization of α6β4 integrin. Primary keratinocytes were seeded on laminin 5-coated glass slides, and keratinocyte cultures were maintained in MEM low in Ca²⁺ for 48 hr. Cultures were then left untreated (A) or infected with PKCα, PKCδ, PKCη or PKCζ recombinant adenoviruses (BE, respectively). Twenty-four hours after infection, keratinocytes were fixed with 4% paraformaldehyde, then gently extracted with 0.2% Triton-X-100, washed with PBS and air-dried as described in the experimental procedure. Cultures were subjected to immunofluorescence analysis with an isotype-specific anti-α6 antibody followed by a FITC-conjugated secondary antibody as described in the experimental procedure.

图9A-B显示了过表达的PKCδ和PKCζ在体外诱导角质化细胞的分离(detachment)。(A)原代角质化细胞不进行处理(C)或者被重组PKCα，δ，η或ζ腺病毒感染。感染后24小时和48小时分析细胞附着，提取细胞并且再次将它们接种到基质涂覆的培养皿上。细胞数目表示为附着细胞的蛋白浓度(mg／个培养皿)。(B)原代角质化细胞不进行处理(C)或者被重组PKCα，δ，η或ζ腺病毒感染。感染后24小时收集培养介质中的脱离的漂浮细胞，对细胞的分离进行分析。细胞数目表示为分离细胞的蛋白浓度(mg／个培养皿)。Figures 9A-B show that overexpressed PKCδ and PKCζ induce keratinocyte detachment in vitro. (A) Primary keratinocytes were left untreated (C) or infected with recombinant PKCα, δ, η or ζ adenovirus. Cell attachment was analyzed 24 hours and 48 hours after infection, cells were extracted and they were replated onto matrix-coated dishes. Cell numbers are expressed as protein concentration (mg/dish) of attached cells. (B) Primary keratinocytes were left untreated (C) or infected with recombinant PKCα, δ, η or ζ adenovirus. Detached floating cells in the culture medium were harvested 24 hours post-infection and analyzed for cell dissociation. Cell numbers are expressed as protein concentration (mg/dish) of isolated cells.

图10证明了在正在积极增殖的角质化细胞中表达PKCη。原代角质化细胞接种(plate)在层粘连蛋白5涂覆的玻璃载玻片上。接种后四十八小时，将角质化细胞与BrdU溶液培育1小时，然后用抗PKCη(红色)和抗BrdU(绿色)抗体进行免疫荧光分析，如下述的实施例部分所述。用共聚焦显微镜对细胞进行扫描，对典型视野进行拍照。Figure 10 demonstrates expression of PKCn in actively proliferating keratinocytes. Primary keratinocytes were plated on laminin 5-coated glass slides. Forty-eight hours after inoculation, keratinocytes were incubated with BrdU solution for 1 hour and then subjected to immunofluorescence analysis using anti-PKCn (red) and anti-BrdU (green) antibodies, as described in the Examples section below. Cells were scanned with a confocal microscope, and typical fields of view were photographed.

图11证明了PKCη诱导，而PKCη突变体减少角质化细胞增殖。原代皮肤角质化细胞被重组PKCη腺病毒或PKCη腺病毒的显性失活突变体(DNPKCη或PKC DNη)感染1小时。感染后四十八小时，进行1小时的³H-胸苷掺入以分析细胞增殖，如下述实施例部分所述。结果表示为cpm／个培养皿。对照是未感染的细胞。Figure 11 demonstrates that PKCn induces and PKCn mutants reduce keratinocyte proliferation. Primary skin keratinocytes were infected with recombinant PKCη adenovirus or a dominant negative mutant of PKCη adenovirus (DNPKCη or PKC DNη) for 1 hr. Forty-eight hours post-infection,³ H-thymidine incorporation was performed for 1 hour to analyze cell proliferation, as described in the Examples section below. Results are expressed as cpm/dish. Controls are uninfected cells.

图12A-B证明了PKCη和DNPKCη的过表达特异调节PKC的定位和细胞形态。原代皮肤角质化细胞被重组PKCη腺病毒或PKCη腺病毒的显性失活突变体(PKC DNη)感染1小时。感染后四十八小时，固定角质化细胞并且(A)进行亮场照相(x20)以及(B)用PKCη特异性抗体然后用FITC结合的二抗进行免疫荧光分析，如实验步骤中所述。对照为未感染的细胞。Figures 12A-B demonstrate that overexpression of PKCn and DNPKCn specifically regulates PKC localization and cell morphology. Primary skin keratinocytes were infected with recombinant PKCη adenovirus or a dominant negative mutant of PKCη adenovirus (PKC DNη) for 1 hr. Forty-eight hours post-infection, keratinocytes were fixed and subjected to (A) bright field photography (x20) and (B) immunofluorescence analysis with PKCn-specific antibody followed by FITC-conjugated secondary antibody as described in the experimental procedure. Controls were uninfected cells.

图13A-B显示了在增殖的角质化细胞中PKCη表达的抑制诱导角质化细胞的分化。原代皮肤角质化细胞在低Ca²⁺的培养基中保持增殖或者在0.12mM Ca²⁺的条件下分化24小时。然后，用重组PKCη腺病毒或PKCη腺病毒的显性失活突变体(PKC DNη)感染角质化细胞1小时。感染后二十四小时，将角质化细胞保持在低Ca²⁺的培养基中或者转至含有0.12mM Ca²⁺的分化培养基中再保持24小时。感染后四十八小时，提取角质化细胞并用SDS-PAGE凝胶分析。用Western blott ing分析PKCη(A)和角蛋白1(B)的表达。Figures 13A-B show that inhibition of PKCn expression in proliferating keratinocytes induces differentiation of keratinocytes. Primary dermal keratinocytes were kept proliferating in low Ca²⁺ medium or differentiated at 0.12 mM Ca²⁺ for 24 hours. Keratinocytes were then infected with recombinant PKCn adenovirus or a dominant negative mutant of PKCn adenovirus (PKC DNn) for 1 hour. Twenty-four hours after infection, keratinocytes were maintained in low Ca2⁺ medium or switched to differentiation medium containing 0.12 mM Ca2⁺ for an additional 24 hours. Forty-eight hours after infection, keratinocytes were extracted and analyzed on SDS-PAGE gels. The expression of PKCη (A) and keratin 1 (B) was analyzed by Western blotting.

图14证明了在小鼠的切割创伤中，体内PKCη的局部表达增加肉芽组织的形成并加速创伤愈合。在裸鼠背部造成7mm的整块皮肤的切口。在发生创伤后1天和4天局部施用对照β-gal，PKCη和PKCα腺病毒悬液。将整块皮肤创伤固定在4％多聚甲醛中，用H&E染色以及亮场显微镜对皮肤切片进行组织学分析。E是表皮，D是真皮。Figure 14 demonstrates that local expression of PKCn in vivo increases granulation tissue formation and accelerates wound healing in excision wounds in mice. A 7 mm skin incision was made on the back of the nude mouse. Control β-gal, PKCη and PKCα adenovirus suspensions were topically applied 1 and 4 days after the occurrence of trauma. Whole skin wounds were fixed in 4% paraformaldehyde, and skin sections were analyzed histologically by H&E staining and bright-field microscopy. E is epidermis and D is dermis.

图15证明了在增殖的角质化细胞中胰岛素，而不是IGF1特异地诱导PKCδ的迁移。分离原代角质化细胞并接种，如下述实施例部分所述。在低Ca²⁺的培养基(0.05mM)中保持增殖的角质化细胞5天，直至它们达到80％汇合。将细胞用10^-7M胰岛素(Ins)或10^-8M IGF1(IGF)刺激15分钟。如上所述溶解细胞，用刺激过的或者未刺激过的对照(Cont)细胞的20μg膜或细胞溶质提取物进行SDS-PAGE并转移。用针对每一个PKC同种型的特异性多克隆抗体对印迹进行探测。Figure 15 demonstrates that insulin, but not IGF1, specifically induces the migration of PKCdelta in proliferating keratinocytes. Primary keratinocytes were isolated and seeded as described in the Examples section below. Proliferating keratinocytes were maintained in low Ca2⁺ medium (0.05 mM) for 5 days until they reached 80% confluency. Cells were stimulated with 10^-7 M insulin (Ins) or 10^-8 M IGF1 (IGF) for 15 minutes. Cells were lysed as described above, subjected to SDS-PAGE and transferred with 20 μg of membrane or cytosolic extracts of stimulated or unstimulated control (Cont) cells. Blots were probed with specific polyclonal antibodies against each PKC isotype.

图16显示了是胰岛素而不是IGF1可诱导PKCδ的活性。为了确定PKCδ的活性，将五天的角质化细胞培养物用10^-7M胰岛素(Ins)或10^-8MIGF1(IGF)刺激指定的时间(1，15，或30分钟)。用特异性抗PKCδ抗体从膜(蓝色条，mem)和细胞溶质(紫色条，cyto)组分中将PKCδ免疫沉淀出来。用体外激酶检测分析PKCδ免疫沉淀物的PKC活性，如实验步骤中所述。每一条表示3次独立实验中3个测定值的平均值±SE。各个值表示为pmol ATP／培养皿／分钟。Figure 16 shows that insulin but not IGF1 induces the activity of PKCdelta. To determine the activity of PKCδ, five-day keratinocyte cultures were stimulated with 10⁻⁷ M insulin (Ins) or 10⁻⁸ MIGF1 (IGF) for the indicated times (1, 15, or 30 minutes). PKCδ was immunoprecipitated from membrane (blue bars, mem) and cytosolic (purple bars, cyto) fractions with specific anti-PKCδ antibodies. PKCδ immunoprecipitates were analyzed for PKC activity using an in vitro kinase assay as described in the experimental procedure. Each bar represents the mean ± SE of 3 determinations in 3 independent experiments. Values are expressed as pmol ATP/dish/min.

图17A-B显示了胰岛素和IGF1对于角质化细胞增殖具有加和效果。增殖的角质化细胞在低Ca²⁺的培养基(0.05mM)中保持5天直至它们达到80％汇合。(A)五天的角质化细胞培养物用指定浓度的胰岛素或IGF1刺激24小时。(B)同时，将角质化细胞用10^-7M胰岛素(Ins)和剂量增加的IGF1(IGF)刺激。在每个浓度，右条(条纹条)表示将两个激素一起加入观察到的增殖。左条证明了10^-7M胰岛素(红色条)和浓度增加的IGF1(灰色条)单独的效果。如实验步骤中所述检测胸苷的掺入。结果代表了六个实验的结果。每一条都是3次测定值的平均值±SE，表示为高于对照的未刺激的角质化细胞的百分数。Figures 17A-B show that insulin and IGF1 have additive effects on keratinocyte proliferation. Proliferated keratinocytes were maintained in low Ca2⁺ medium (0.05 mM) for 5 days until they reached 80% confluency. (A) Five-day keratinocyte cultures were stimulated for 24 hours with the indicated concentrations of insulin or IGF1. (B) Simultaneously, keratinocytes were stimulated with 10⁻⁷ M insulin (Ins) and increasing doses of IGF1 (IGF). At each concentration, the right bar (striped bar) represents the proliferation observed when both hormones were added together. The left bar demonstrates the effect of 10^-7 M insulin (red bar) and increasing concentrations of IGF1 (gray bar) alone. Incorporation of thymidine was detected as described in the experimental procedure. Results represent the results of six experiments. Each bar is mean ± SE of 3 determinations expressed as percentage of unstimulated keratinocytes above control.

图18A-B证明了重组PKC腺病毒构建体的过表达。用含有野生型PKCδ(WTPKCδ)，野生型PKCα(WTPKCα)，或显性失活PKCδ突变体(DNPKCδ)的重组腺病毒构建体感染角质化细胞培养物。(A)感染后将细胞培养24小时，收集，对20μg蛋白质提取物用特异性抗PKCα或抗PKCδ抗体进行Western blotting分析。印迹结果代表了5次独立实验的结果。(B)感染后二十四个小时，收集细胞，用体外激酶检测分析PKCα或PKCδ免疫沉淀物。Figures 18A-B demonstrate overexpression of recombinant PKC adenoviral constructs. Keratinocyte cultures were infected with recombinant adenoviral constructs containing wild-type PKCδ (WTPKCδ), wild-type PKCα (WTPKCα), or a dominant negative PKCδ mutant (DNPKCδ). (A) Cells were cultured for 24 hours after infection, collected, and 20 μg of protein extracts were analyzed by Western blotting with specific anti-PKCα or anti-PKCδ antibodies. Blot results represent the results of 5 independent experiments. (B) Twenty-four hours after infection, cells were collected and immunoprecipitates were analyzed for PKCα or PKCδ immunoprecipitates using in vitro kinase assays.

图19显示了PKC过表达对于胰岛素或IGF1诱导的增殖的作用。未感染的(浅蓝条)，或过表达WTPKCδ(深蓝条)或DNPKCδ(斜线蓝条)的细胞用10^-7M胰岛素(Ins)，10^-8M IGF1(IGF)或二者(Ins+IGF)处理24小时。如实验步骤中所述检测胸苷的掺入。每一条表示用单独的培养物进行的3次实验的3个测定值的平均值±SE。各个值表示为基于作为对照的每个实验中同一培养物的未刺激的角质化细胞的百分数。Figure 19 shows the effect of PKC overexpression on insulin or IGF1 induced proliferation. Uninfected (light blue bars), or cells overexpressing WTPKCδ (dark blue bars) or DNPKCδ (oblique blue bars) were treated with 10^-7 M insulin (Ins), 10^-8 M IGF1 (IGF) or both (Ins +IGF) for 24 hours. Incorporation of thymidine was detected as described in the experimental procedure. Each bar represents the mean ± SE of 3 determinations from 3 experiments performed with separate cultures. Values are expressed as a percentage of unstimulated keratinocytes from the same culture in each experiment as a control.

图20显示了PKCδ活性的抑制特异性消除胰岛素诱导的角质化细胞的增殖。如下述实施例部分所述培养原代角质化细胞。未感染的细胞或DNPKCδ感染的角质化细胞用下述浓度的生长因子刺激24小时：10^-7M胰岛素(Ins)，10^-8M IGF1(IGF)，10ng／ml EGF，10ng／ml PDGF，1ng／ml KGF或5ng／ml ECGF。如下述实施例部分所述检测胸苷的掺入。每一条都表示用单独的培养物进行的3次实验的3个测定值的平均值±SE。各个值表示为基于作为对照的每个实验中同一培养物的未刺激的角质化细胞的百分数。Figure 20 shows that inhibition of PKCδ activity specifically abolishes insulin-induced proliferation of keratinocytes. Primary keratinocytes were cultured as described in the Examples section below. Uninfected cells or DNPKCδ-infected keratinocytes were stimulated for 24 hours with growth factors at the following concentrations: 10^-7 M insulin (Ins), 10^-8 M IGF1 (IGF), 10 ng/ml EGF, 10 ng/ml PDGF, 1ng/ml KGF or 5ng/ml ECGF. Incorporation of thymidine was detected as described in the Examples section below. Each bar represents the mean ± SE of 3 determinations from 3 experiments performed with separate cultures. Values are expressed as a percentage of unstimulated keratinocytes from the same culture in each experiment as a control.

图21显示了过表达的PKCδ特异调节胰岛素诱导的角质化细胞增殖。原代角质化细胞如图1所述那样培养。未感染的细胞或用过表达的WTPKCδ感染的角质化细胞用下述浓度的生长因子刺激24小时：10^-7M胰岛素(Ins)，10^-8M IGF1(IGF)，10ng／ml EGF，10ng／ml PDGF，1ng／ml KGF或5ng／ml ECGF。如下述实施例部分所述检测胸苷的掺入。每一条都表示用单独的培养物进行的3次实验的3个测定值的平均值±SE。各个值表示为基于作为对照的每个实验中同一培养物的未刺激的角质化细胞的百分数。Figure 21 shows that overexpressed PKCδ specifically regulates insulin-induced keratinocyte proliferation. Primary keratinocytes were cultured as described in Figure 1. Uninfected cells or keratinocytes infected with overexpressed WTPKCδ were stimulated for 24 hours with the following concentrations of growth factors: 10^-7 M insulin (Ins), 10^-8 M IGF1 (IGF), 10 ng/ml EGF, 10 ng /ml PDGF, 1ng/ml KGF or 5ng/ml ECGF. Incorporation of thymidine was detected as described in the Examples section below. Each bar represents the mean ± SE of 3 determinations from 3 experiments performed with separate cultures. Values are expressed as a percentage of unstimulated keratinocytes from the same culture in each experiment as a control.

图22A-B证实了PKCδ和PKCζ在体内皮肤创伤愈合过程中具有重要作用。使用新开发的同种型特异性PKC无效小鼠的体内小鼠模型，对PKCα，PKCδ和PKCζ无效小鼠和它们的野生型的同窝出生小鼠进行创伤愈合研究。麻醉小鼠并在小鼠背上通过穿刺活检造成直径4mm的皮肤。在进行一周的跟踪后取下小鼠皮肤，用破裂室方法对皮肤薄片进行创伤强度测试以确定皮肤创伤的愈合。其值表达为破裂压力，其代表在监测室内直至发生破裂的最大压力。结果为12-20只小鼠的不同组的测定值。实验重复至少3次。Figures 22A-B demonstrate that PKCδ and PKCζ play an important role in skin wound healing in vivo. Wound healing studies were performed in PKCα, PKCδ, and PKCζ-null mice and their wild-type littermates using a newly developed in vivo mouse model of isoform-specific PKC-null mice. Mice were anesthetized and a 4 mm diameter skin was created by punch biopsy on the back of the mouse. Mouse skin was removed after one week of follow-up, and skin slices were tested for wound strength using the rupture chamber method to determine skin wound healing. Its value is expressed as burst pressure, which represents the maximum pressure in the monitoring chamber until rupture occurs. Results are measurements of different groups of 12-20 mice. Experiments were repeated at least 3 times.

图23确定了原代皮肤角质化细胞中STAT3和PKCδ之间的特异的相互作用。原代角质化细胞不进行处理(上)或者用同种型特异性重组PKC腺病毒感染1小时(下)。提取细胞用同种型特异性PKC抗体进行免疫沉淀(IP)。用抗PKC或抗STAT3抗体对免疫沉淀物进行Western blot分析。Figure 23 identifies the specific interaction between STAT3 and PKCδ in primary skin keratinocytes. Primary keratinocytes were left untreated (top) or infected with isotype-specific recombinant PKC adenovirus for 1 hour (bottom). Extracted cells were immunoprecipitated (IP) with an isotype-specific PKC antibody. Immunoprecipitates were analyzed by Western blot with anti-PKC or anti-STAT3 antibodies.

图24证明了PKCδ的活化对于胰岛素诱导的STAT3的转录活化具有重要作用。将原代角质化细胞接种在玻璃载玻片上，在低Ca⁺⁺(0.05mmol／l)的培养基中保持5天直至它们达到80％汇合。细胞不进行处理(Cont，上)或者用5μM Rottlerin预处理7分钟(R，下)，然后用10^-7M胰岛素处理5分钟(Ins)。用甲醇固定细胞，洗涤并风干。用抗磷酸-Tyr-705-STAT3抗体然后用FITC结合的二抗免疫荧光以分析培养物。用共聚焦显微镜扫描细胞。Figure 24 demonstrates that the activation of PKCδ plays an important role in the insulin-induced transcriptional activation of STAT3. Primary keratinocytes were seeded on glass slides and maintained in low Ca⁺⁺ (0.05 mmol/l) medium for 5 days until they reached 80% confluency. Cells were left untreated (Cont, top) or pretreated with 5 μM Rottlerin for 7 minutes (R, bottom) and then treated with 10⁻⁷ M insulin for 5 minutes (Ins). Cells were fixed with methanol, washed and air-dried. Cultures were analyzed by immunofluorescence with anti-phospho-Tyr-705-STAT3 antibody followed by FITC-conjugated secondary antibody. Scan cells with a confocal microscope.

图25证明了过表达DN PKCδ抑制PKCδ和STAT3的过表达诱导的角质化细胞的增殖。原代角质化细胞用含有β-Gal(作为对照)，PKCδ，WT STAT3，DN STAT3的重组腺病毒构建体感染1小时或用DN PKCδ，然后用STAT3进行双重感染。感染后24小时，进行1小时的³H-胸苷掺入以分析细胞增殖。结果表示为DPM／mg蛋白质。每一条都是用同一培养物的平板(plate)进行的三次测定的平均值。Figure 25 demonstrates that overexpression of DN PKCδ inhibits proliferation of keratinocytes induced by overexpression of PKCδ and STAT3. Primary keratinocytes were infected with a recombinant adenoviral construct containing β-Gal (as a control), PKCδ, WT STAT3, DN STAT3 for 1 hour or double-infected with DN PKCδ followed by STAT3. 24 hours post-infection,³ H-thymidine incorporation was performed for 1 hour to analyze cell proliferation. Results are expressed as DPM/mg protein. Each bar is the mean of three determinations performed on plates of the same culture.

图26证明了胰岛素施用的浓度和频率对于体内创伤愈合的作用。在8-10周龄的C57BL小鼠背部切出创伤切口，用PBS(对照)处理或者以不同浓度和频率进行胰岛素处理(也就是七天里每天重复施用相对于单独施用一次)。致创后七天将小鼠处死，检测处理的创伤部位。结果表示为mm²创伤部位，每一条都是六次重复的平均值±标准差(p<0.005)。Figure 26 demonstrates the effect of concentration and frequency of insulin administration on wound healing in vivo. Trauma incisions were made on the back of 8-10 week old C57BL mice and treated with PBS (control) or insulin at different concentrations and frequencies (ie repeated daily administration for seven days versus once alone). Mice were sacrificed seven days after wounding and the treated wound sites were examined. Results are expressed as^mm2 wound site, and each bar is the mean±SD of six replicates (p<0.005).

图27证明了胰岛素施用的浓度和频率对于体内创伤愈合的组织学效果。在8-10周龄的C57BL小鼠背部切出创伤切口，以不同浓度和频率进行胰岛素处理(也就是七天里每天重复施用相对于单独施用一次)。致创后七天制备组织学创伤切片，分析表皮和真皮的闭合(创伤收缩)。表皮的闭合用Keratin14(K14)抗体染色(左)检测，如果创伤的整个裂口都是染色阳性，则视为阳性。如果真皮创伤的两边在光学显微镜下在x100的放大倍数下在一个视野中都能看见(右)，则真皮的闭合视为阳性。结果表示为创伤闭合相对于对照的百分数，每一条都是六次重复的平均值。Figure 27 demonstrates the histological effect of concentration and frequency of insulin administration on wound healing in vivo. Traumatic incisions were made on the back of 8-10 week old C57BL mice and insulin treatments were administered at different concentrations and frequencies (ie, repeated daily versus single administration for seven days). Histological wound sections were prepared seven days after wounding and analyzed for closure of the epidermis and dermis (wound contraction). Closure of the epidermis was detected with Keratin 14 (K14) antibody staining (left), and was considered positive if the entire breach of the wound stained positively. Closure of the dermis is considered positive if both sides of the dermal wound are visible in one field under a light microscope at x100 magnification (right). Results are expressed as percent wound closure relative to control and each bar is the mean of six replicates.

图28证明了胰岛素和血小板衍生生长因子(platelet derivedgrowth factor，PDGF-BB)组合使用对于体内创伤愈合的协同作用。在8-10周龄的C57BL小鼠背部切出创伤切口，用胰岛素，PDGF-BB，或组合使用胰岛素和PDGF-BB进行单次施用。致创后七天处死小鼠，取活组织进行表皮和真皮闭合(创伤收缩)的组织学分析。表皮的闭合用Keratin14(K14)抗体染色(左)检测，如果创伤的整个裂口都是染色阳性，则视为阳性。如果真皮创伤的两边在光学显微镜下在x100的放大倍数下在一个视野中都能看见(右)，则真皮的闭合视为阳性。结果表示为条形图，为创伤闭合相对于对照的百分数，每一条都是六次重复的平均值。Figure 28 demonstrates the synergistic effect of insulin and platelet derived growth factor (platelet derived growth factor, PDGF-BB) combination on wound healing in vivo. A traumatic incision was made on the back of 8-10 week old C57BL mice and a single administration of insulin, PDGF-BB, or a combination of insulin and PDGF-BB was performed. Mice were sacrificed seven days after wounding and biopsies were taken for histological analysis of epidermal and dermal closure (wound contraction). Closure of the epidermis was detected with Keratin 14 (K14) antibody staining (left), and was considered positive if the entire breach of the wound stained positively. Closure of the dermis is considered positive if both sides of the dermal wound are visible in one field under a light microscope at x100 magnification (right). Results are presented as a bar graph as percent wound closure relative to control, each bar is the mean of six replicates.

图29A-D是示出胰岛素和PKCα抑制剂组合使用对体内创伤愈合的形态学作用的照片。在8-10周龄的C57BL小鼠背部切出创伤切口，不进行处理(对照)或用胰岛素(HO／01)和PKCα抑制剂(HO／02)组合处理。致创后7天除下皮肤活组织进行形态学观察。图29A-B显示了对照创伤，图29C-D显示了处理的创伤。29A-D are photographs showing the morphological effects of insulin and a PKCα inhibitor combination on wound healing in vivo. Trauma incisions were made on the back of 8-10 week old C57BL mice, either untreated (control) or treated with a combination of insulin (HO/01) and a PKCα inhibitor (HO/02). Seven days after the injury, the skin biopsies were removed for morphological observation. Figures 29A-B show control wounds and Figures 29C-D show treated wounds.

图30是示出胰岛素和PKCα抑制剂组合使用对真皮闭合(创伤收缩)的作用的组织显微照片。在8-10周龄的C57BL小鼠背部切出创伤切口，不进行处理(对照)或用胰岛素(HO／01)和PKCα抑制剂(HO／02)组合起来每天处理。致创后七天处死处理的小鼠。制作组织学创伤切片并在光学显微镜下观察。如果真皮创伤的两边在x100的放大倍数下在一个视野中都能看见，则真皮的闭合视为阳性。未处理的对照切片(左)的张开的创伤区域太大不能容纳于一个x100放大的视野中，而处理的创伤切片(右)表现出真皮闭合阳性。黄色斑点线标出了真皮的边缘。Figure 30 is a micrograph of tissue showing the effect of the combination of insulin and a PKCa inhibitor on dermal closure (wound contraction). Trauma incisions were made on the back of 8-10 week old C57BL mice, either untreated (control) or treated daily with a combination of insulin (HO/01) and a PKCα inhibitor (HO/02). Treated mice were sacrificed seven days after wounding. Histological wound sections were made and observed under a light microscope. Closure of the dermis is considered positive if both sides of the dermal wound are visible in one field at x100 magnification. Untreated control slices (left) had a distended wound area that was too large to fit in a x100 magnified field of view, while treated wound slices (right) showed positive dermal closure. A line of yellow spots marks the edge of the dermis.

图31是示出胰岛素和PKCα抑制剂组合使用对表皮闭合的作用的组织显微照片。在8-10周龄的C57BL小鼠背部切出创伤切口，不进行处理或用胰岛素(HO／01)和PKCα抑制剂(HO／02)组合起来每天处理。致创后七天处死处理的小鼠。制备创伤的组织切片，用角蛋白14(指示基底角质化细胞)染色，在光学显微镜下观察。未处理的对照切片(左)中的张开的创伤区域(箭头标出)太大不能容纳于一个x100放大的视野中，而处理的创伤切片(右)整个裂口表现出表皮闭合。Figure 31 is a micrograph of tissue showing the effect of the combination of insulin and a PKCa inhibitor on epidermal closure. Traumatic incisions were made on the back of 8-10 week old C57BL mice, which were left untreated or treated daily with a combination of insulin (HO/01) and a PKCα inhibitor (HO/02). Treated mice were sacrificed seven days after wounding. Wounded tissue sections were prepared, stained with keratin 14 (indicating basal keratinocytes), and observed under a light microscope. The open wound area (indicated by the arrow) in the untreated control section (left) is too large to fit in a x100 magnified field of view, while the treated wound section (right) exhibits epidermal closure of the entire tear.

图32是示出胰岛素和PKCα抑制剂组合使用对表皮细胞空间分化的作用的组织显微照片。致创小鼠(C57BL，8-10周龄)用胰岛素(HO／01)和PKCα抑制剂(HO／02)组合起来每天局部施用进行处理。致创后七天处死处理的小鼠。制备创伤的组织切片，用突出显示细胞空间分化的初始阶段的keratin1(K1)抗体染色。未处理的对照切片(左)表现出大的未分化的创伤区域(由箭头标出)，而处理的创伤切片(右)中则观察到大量的表皮重建。Figure 32 is a micrograph of tissue showing the effect of the combination of insulin and a PKC[alpha] inhibitor on the spatial differentiation of epidermal cells. Wounded mice (C57BL, 8-10 weeks old) were treated with a combination of insulin (HO/01) and a PKCα inhibitor (HO/02) administered topically daily. Treated mice were sacrificed seven days after wounding. Wounded tissue sections were prepared and stained with an antibody to keratin1 (K1) that highlights the initial stages of cellular spatial differentiation. Untreated control sections (left) exhibit large undifferentiated wound areas (marked by arrows), whereas extensive epidermal remodeling is observed in treated wound sections (right).

图33证明了胰岛素和PKCα抑制剂组合使用对体内创伤愈合的定量作用。致创小鼠(C57BL，8-10周龄)用胰岛素(HO／01)和PKCα抑制剂(HO／02)组合起来每天局部施用进行处理。致创后七天处死处理的小鼠。制备创伤的组织切片，进行真皮收缩，表皮闭合和空间分化的分析，如上述图30-32所述。条形图显示出了通过组织学分析得出的每一个处理组中完全愈合的创伤的发生率(百分数)。Figure 33 demonstrates the quantitative effect of the combination of insulin and a PKCα inhibitor on wound healing in vivo. Wounded mice (C57BL, 8-10 weeks old) were treated with a combination of insulin (HO/01) and a PKCα inhibitor (HO/02) administered topically daily. Treated mice were sacrificed seven days after wounding. Histological sections of wounds were prepared for analysis of dermal contraction, epidermal closure and spatial differentiation, as described above for Figures 30-32. Bar graphs show the incidence (percentage) of fully healed wounds in each treatment group by histological analysis.

图34A-G是真皮细胞中PKCα的表达和／或活性的抑制和另一种PKC同种型的表达和／或活性的调节，或者给真皮细胞施用激素对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤成纤维细胞用显性失活(DN)的激酶失活的PKCα感染。二十四小时后实施刮擦，培养物不进行处理(图34A)，或者用野生型(WT)PKCδ(图34B)，PKCη(图34C)，WT PKCζ(图34D)或WT PKCε(图34E)感染。另外，PKCα抑制的培养物用adipsin(2μg／ml；图34F)或胰岛素(6.7x10^-7M；图34G)处理。处理后24小时拍照。34A-G are graphs of the combined effects of inhibition of PKCα expression and/or activity and modulation of another PKC isoform expression and/or activity in dermal cells, or administration of hormones to dermal cells, on closure of skin wounds in vitro. Example photo. Cultured primary skin fibroblasts were infected with dominant negative (DN) kinase-inactive PKCα. Twenty-four hours later, scraping was performed and cultures were left untreated (FIG. 34A) or treated with wild-type (WT) PKCδ (FIG. 34B), PKCη (FIG. 34C), WT PKCζ (FIG. 34D) or WT PKCε (FIG. 34E). )Infect. Additionally, PKCα-inhibited cultures were treated with adipsin (2 μg/ml; FIG. 34F ) or insulin (6.7×10⁻⁷ M; FIG. 34G ). Photographs were taken 24 hours after treatment.

图35A-H是真皮细胞中PKCα的表达和／或活性的抑制和另一种PKC同种型的表达和／或活性的调节，或者给真皮细胞施用生长因子对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤成纤维细胞用显性失活(DN)的激酶失活的PKCα感染。二十四小时后实施刮擦，培养物不进行处理(图35A)，或者用野生型(WT)PKCε(图35D)，WT PKCζ(图35E)或WT PKCη(图35F)感染。或者，PKCα抑制的培养物用IL-6(每个培养血1μg；图35B)，KGF(每个培养皿1μg；图35C)，PKCδRACK(10^-7M；图35H)或TNFα(12μg／ml；图35G)处理。处理后24小时拍照。Figures 35A-H are the combined effects of inhibition of PKCα expression and/or activity and modulation of another PKC isoform expression and/or activity in dermal cells, or administration of growth factors to dermal cells, on closure of skin wounds in vitro sample photo of . Cultured primary skin fibroblasts were infected with dominant negative (DN) kinase-inactive PKCα. Twenty-four hours later scraping was performed and cultures were left untreated (FIG. 35A) or infected with wild-type (WT) PKCε (FIG. 35D), WT PKCζ (FIG. 35E) or WT PKCη (FIG. 35F). Alternatively, PKCα-inhibited cultures were treated with IL-6 (1 μg per culture; Figure 35B), KGF (1 μg per culture dish; Figure 35C), PKCδRACK (10^-7 M; Figure 35H ) or TNFα (12 μg/ml ; FIG. 35G ) treatment. Photographs were taken 24 hours after treatment.

图36A-B是真皮细胞中PKCζ的表达和／或活性的抑制，和给真皮细胞施用生长因子对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤成纤维细胞用显性失活(DN)的激酶失活的PKCζ(DNζ)感染。二十四小时后实施刮擦，培养物不进行处理(图36A)，或者用KGF(每个培养皿1μg；图36B)处理。处理后24小时拍照。36A-B are photographs illustrating the combined effect of inhibition of PKCζ expression and/or activity in dermal cells, and administration of growth factors to dermal cells on closure of skin wounds in vitro. Cultured primary skin fibroblasts were infected with dominant negative (DN) kinase-inactive PKCζ (DNζ). Twenty-four hours later scraping was performed and cultures were left untreated (Figure 36A) or treated with KGF (1 μg per dish; Figure 36B). Photographs were taken 24 hours after treatment.

图37A-D是真皮细胞中PKCζ的表达和／或活性的抑制，和给真皮细胞施用生长因子或激素对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤角质化细胞用显性失活(DN)的激酶失活的PKCζ(DNζ)感染。二十四小时后实施刮擦，培养物不进行处理(图37A)，或者用IL-6(每个培养皿1μg；图37B)，TNFα(12μg／ml；图37C)或adiponectin(每个培养皿1μg；图37D)处理。处理后24小时拍照。37A-D are photographs illustrating the combined effect of inhibition of PKCζ expression and/or activity in dermal cells and administration of growth factors or hormones to dermal cells on closure of skin wounds in vitro. Cultured primary skin keratinocytes were infected with dominant negative (DN) kinase-inactive PKCζ (DNζ). Twenty-four hours later the scraping was performed and the cultures were left untreated (Figure 37A) or treated with IL-6 (1 μg per culture dish; Figure 37B), TNFα (12 μg/ml; Figure 37C) or adiponectin (perculture dish dish 1 μg; FIG. 37D ). Photographs were taken 24 hours after treatment.

图38A-E是真皮细胞中PKCβ的表达和／或活性的抑制，和给真皮细胞施用生长因子，胰岛素或GW9662对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤成纤维细胞用显性失活(DN)的激酶失活形式的PKCβ(DNβ)感染。二十四小时后实施刮擦，培养物不进行处理(图38A)，或者用KFG(每个培养皿1μg；图38B)，IL-6(每个培养皿1μg；图38C)，胰岛素(6.7x10^-7M；图38D)或GW9662(每个培养皿1μg；图38E)处理。处理后24小时拍照。38A-E are photographs illustrating the combined effect of inhibition of PKCβ expression and/or activity in dermal cells and administration of growth factors, insulin or GW9662 to dermal cells on closure of skin wounds in vitro. Cultured primary skin fibroblasts were infected with the dominant negative (DN) kinase-inactive form of PKCβ (DNβ). Twenty-four hours later the scraping was performed and the cultures were left untreated (Figure 38A) or treated with KFG (1 μg per dish; Figure 38B), IL-6 (1 μg per dish; Figure 38C), insulin (6.7 x10⁻⁷ M; FIG. 38D ) or GW9662 (1 μg per dish; FIG. 38E ) treatment. Photographs were taken 24 hours after treatment.

图39A-E是真皮细胞中PKCδ的表达和／或活性的抑制和另一种PKC同种型的表达和／或活性的调节，或者给真皮细胞施用激素对于体外皮肤创伤的闭合的组合效果的示例照片。将培养的原代皮肤角质化细胞用野生型(WT)的激酶形式的PKCδ(DNδ)感染。二十四小时后实施刮擦，培养物不进行处理(图39A)，或者用WT PKCζ(PKCζ；图39B)，WT PKCε(PKCε；图39C)或DN PKCα(PKCα；图39D)感染。或者，PKCδ提高的培养物用adipsin(2μg／ml；图39E)处理。处理后48小时拍照。39A-E are graphs of the combined effects of inhibition of PKCδ expression and/or activity and modulation of another PKC isoform expression and/or activity in dermal cells, or administration of hormones to dermal cells, on closure of skin wounds in vitro. Example photo. Cultured primary skin keratinocytes were infected with the wild-type (WT) kinase form of PKCδ (DNδ). Twenty-four hours later scraping was performed and cultures were left untreated (Figure 39A) or infected with WT PKCζ (PKCζ; Figure 39B), WT PKCε (PKCε; Figure 39C) or DN PKCα (PKCα; Figure 39D). Alternatively, cultures with elevated PKCδ were treated with adipsin (2 μg/ml; FIG. 39E ). Photographs were taken 48 hours after treatment.

图40A-F是施用共聚物-1，胰岛素，PKCα伪底物，或其组合对于体外皮肤创伤闭合的效果的示例照片。培养的原代皮肤角质化细胞不进行处理(图40A)，或者仅用胰岛素(6.7x10^-7M；图40B)，仅用共聚物-1(55μg／培养皿；图40C)，胰岛素和PKCα伪底物的混合物(分别为6.7x10^-7M和10⁷M；图40D)，共聚物-1和胰岛素的混合物(分别为55μg／培养皿和6.7x10^-7M；图40E)，或共聚物-1，胰岛素和PKCα伪底物的混合物(分别为55μg／培养皿，6.7x10^-7M和10⁷M；图40F)处理。处理后48小时拍照。40A-F are photographs illustrating the effect of administration of Copolymer-1, insulin, a PKCα pseudosubstrate, or a combination thereof, on skin wound closure in vitro. Cultured primary dermal keratinocytes were left untreated (Fig. 40A), or treated with insulin alone (6.7x10^-7 M; Fig. 40B ), only with copolymer-1 (55 μg/dish; Fig. 40C ), insulin and PKCα Mixtures of pseudosubstrates (6.7x10^-7 M and 10⁷ M, respectively; Fig. 40D ), mixtures of copolymer-1 and insulin (55 μg/dish and 6.7x10^-7 M, respectively; Fig. 40E ), or copolymer Substance-1, a mixture of insulin and PKCα pseudo-substrates (55 μg/dish, 6.7×^{10 −7} M and 10⁷ M, respectively; FIG. 40F ). Photographs were taken 48 hours after treatment.

图41A-D是共聚物-1，胰岛素，PKCα伪底物，或其组合对于体内创伤愈合的效果的示例照片。致创小鼠不进行处理(图41A)或在4天里每天局部施用共聚物-1(55μg／ml；图41B)，共聚物-1和胰岛素的混合物(分别为55μg／皿l和1μM；图41C)，或共聚物-1，胰岛素和PKCα伪底物的混合物(分别为55μg／ml，1μM和1μM；图41D)。致创后4天拍照。41A-D are photographs illustrating the effect of Copolymer-1, insulin, PKCα pseudo-substrates, or a combination thereof on wound healing in vivo. Wounded mice were left untreated (Fig. 41A) or were topically administered daily for 4 days with Copolymer-1 (55 μg/ml; Fig. 41B), a mixture of Copolymer-1 and insulin (55 μg/ml and 1 μM, respectively; FIG. 41C ), or mixtures of copolymer-1, insulin and PKCα pseudosubstrates (55 μg/ml, 1 μM and 1 μM, respectively; FIG. 41D ). Photo taken 4 days after trauma.

图42A-H是接近创伤裂口的胸腺对于创伤愈合过程的效果的示例的组织显微照片。图42A-B显示了正常的成年啮齿动物的胸腺的200倍(x200)放大图。图42C显示了7天龄创伤的40倍(x40)放大图，在离创伤裂口非常接近的地方观察到胸腺(红色正方形，放大200倍，图42D)。创伤重新形成上皮，形成肉芽组织，真皮正在进行收缩。图42E-F显示了放大40倍(图42E)和200倍(图42F)的STZ糖尿病小鼠的9天龄创伤，在创伤裂口附近没有观察到胸腺，没有观察到重新形成上皮，肉芽组织，或真皮收缩。图42G显示了放大40倍的STZ糖尿病小鼠的9天龄创伤。将创伤用胰岛素和PKCα伪底物的混合物处理。在创伤裂口附近观察到胸腺(红色正方形，放大20倍，图42H)。创伤重新形成上皮，形成肉芽组织，真皮正在进行收缩。Figures 42A-H are tissue micrographs illustrating the effect of the thymus proximal to the wound breach on the wound healing process. Figures 42A-B show 200-fold (x200) magnifications of a normal adult rodent thymus. Figure 42C shows a 40X (x40) magnification of a 7 day old wound, where the thymus was observed very close to the wound opening (red squares, 200X magnification, Figure 42D). The wound is re-epithelialized, granulation tissue is formed, and the dermis is shrinking. Figure 42E-F shows 9-day-old wounds of STZ diabetic mice magnified 40 times (Figure 42E) and 200 times (Figure 42F), no thymus was observed near the wound gap, no re-epithelialization, granulation tissue was observed, or dermal shrinkage. Figure 42G shows a 9-day-old wound of an STZ diabetic mouse at 40X magnification. Wounds were treated with a mixture of insulin and PKCα pseudo-substrates. The thymus (red square, 20X magnification, Figure 42H) was observed near the wound breach. The wound is re-epithelialized, granulation tissue is formed, and the dermis is shrinking.

图43是胰岛素和PKCα抑制剂组合使用对创伤和损伤皮肤愈合的作用的示例照片。在Large Whites&Landrace家养猪的背部形成纵向创伤切口，在15天中每天用PBS(对照)或1μM胰岛素和1μMPKCα伪底物(HO／03／03)的混合物处理。致创后30天对创伤拍照。与缓冲液对照相比，HO／03／03处理的创伤完全愈合，没有疤痕形成，显著增加了皮肤的美感。Figure 43 is a photograph illustrating the effect of the combination of insulin and a PKCα inhibitor on wound and damaged skin healing. Longitudinal wound incisions were made on the back of Large Whites & Landrace domestic pigs and treated daily for 15 days with either PBS (control) or a mixture of 1 μΜ insulin and 1 μΜ MPKCα pseudo-substrate (HO/03/03). Wounds were photographed 30 days after trauma. Compared with the buffer control, HO/03/03 treated wounds healed completely without scar formation and significantly increased the aesthetics of the skin.

优选实施方案preferred embodiment

本发明是用于调节丝氨酸／苏氨酸蛋白激酶，也称为PKC的表达和／或活化，以诱导和／或加速细胞增殖和／或细胞分化，从而加速创伤的愈合过程的方法和药物组合物。根据本发明的教导，这种表达的调节可以通过下述作用实现：(i)用PKC表达构建体转化创伤细胞；(ii)用顺式作用元件转化创伤细胞，该元件将被插入到创伤细胞内源PKC基因上游邻近位置；(iii)施用胰岛素和其它能与胰岛素协同作用的试剂以调节创伤细胞中PKC的表达和／或活化；(iv)用胰岛素表达构建体转化创伤细胞，其产生的表达的和分泌的胰岛素可作为PKC的表达和／或活化的上调剂；(v)用顺式作用元件转化创伤细胞，该元件将被插入到创伤细胞内源胰岛素基因上游邻近位置，其产生的表达的和分泌的胰岛素可作为PKC的表达和／或活化的上调剂；(vi)向创伤中植入胰岛素分泌细胞；(vii)用反式作用因子，例如PDX1转化创伤细胞，以诱导内源胰岛素的产生和分泌，胰岛素可作为PKC的表达和／或活化的上调剂；以及(viii)给创伤施用PKC调节剂。The present invention is a method and drug combination for regulating the expression and/or activation of serine/threonine protein kinase, also known as PKC, to induce and/or accelerate cell proliferation and/or cell differentiation, thereby accelerating the healing process of wounds things. According to the teachings of the present invention, the regulation of this expression can be achieved by: (i) transforming wounding cells with a PKC expression construct; (ii) transforming wounding cells with a cis-acting element, which will be inserted into the wounding cells (iii) administration of insulin and other agents that can act synergistically with insulin to regulate expression and/or activation of PKC in wounded cells; (iv) transformed wounded cells with an insulin expression construct that produces Expressed and secreted insulin can act as an upregulator of PKC expression and/or activation; (v) transformation of wounded cells with a cis-acting element that will be inserted adjacent to the upstream endogenous insulin gene of wounded cells, which produces Expressed and secreted insulin acts as an upregulator of PKC expression and/or activation; (vi) implantation of insulin-secreting cells into the wound; (vii) transformation of wounded cells with trans-acting factors, such as PDX1, to induce endogenous production and secretion of insulin, which may act as an upregulator of PKC expression and/or activation; and (viii) administering a PKC modulator to the wound.

参考附图以及说明书可更好地理解本发明所述方法和药物组合物的原理和操作。The principles and operation of the methods and pharmaceutical compositions of the present invention may be better understood with reference to the drawings and description.

在详细描述本发明的至少一个实施方案之前，应当理解本发明不限于下述说明书中或实施例部分例示的具体的组分组成或排列。本发明能够以其它实施方案或者以不同的方式实施。而且，应当理解本文所使用的措辞和术语仅是为了描述使用，而不应当视为是限制。Before at least one embodiment of the present invention is described in detail, it is to be understood that this invention is not limited to the specific compositions or arrangements of components exemplified in the following specification or in the Examples section. The invention is capable of other embodiments or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

成年人皮肤包括两层：角质化的成层的表皮层和在下面的厚层的富含胶原的真皮层结缔组织，提供支持和营养。皮肤作为保护屏障抵御外界。因此任何皮肤的损伤或破裂都必须被快速有效地修复。如上述背景技术部分所述，修复的第一个阶段是通过形成血液凝块堵住最初的创伤来实现的。然后炎性细胞，成纤维细胞和毛细血管侵入血液凝块形成肉芽组织。后续阶段包括创伤的上皮重建，其中基底角质化细胞必须失去与半桥粒的连接，角质化细胞迁移到肉芽组织上，覆盖住创伤。角质化细胞迁移以后，角质化细胞加速增殖，其容许代替在损伤中损失的细胞。在创伤被单层的角质化细胞覆盖之后，形成新成层的表皮，重建新的基底膜(20-23)。已证明几种生长因子参与该过程，包括生长因子的EGF家族，KGF，PDGF和TGFβ1(22-24)。在这些生长因子中，EGF和KGF与表皮角质化细胞的增殖和迁移的调节密切相关(25，26)。对于了解创伤愈合生物学而言很重要的是了解触发创伤中的细胞迁移、增殖、并在创伤裂口上铺放新的基质的信号。Adult skin consists of two layers: the keratinized, stratified epidermis and the underlying thick, collagen-rich dermis, which provides support and nourishment. The skin acts as a protective barrier against the outside world. Therefore any damage or break in the skin must be repaired quickly and efficiently. As mentioned in the background section above, the first stage of repair is achieved by the formation of a blood clot that plugs the original wound. Inflammatory cells, fibroblasts and capillaries then invade the blood clot to form granulation tissue. Subsequent stages include reepithelialization of the wound, in which basal keratinocytes must lose their connection to the hemidesmosomes, and keratinocytes migrate onto the granulation tissue, covering the wound. Following keratinocyte migration, keratinocyte proliferation is accelerated, which allows replacement of cells lost in injury. After the wound is covered with a monolayer of keratinocytes, a new stratified epidermis forms, remodeling a new basement membrane (20-23). Several growth factors have been shown to be involved in this process, including the EGF family of growth factors, KGF, PDGF and TGFβ1 (22-24). Among these growth factors, EGF and KGF are closely related to the regulation of proliferation and migration of epidermal keratinocytes (25, 26). Important to understanding the biology of wound healing is the understanding of the signals that trigger cell migration, proliferation, and deposition of new matrix in wound wounds in wounds.

为了便于理解下面公开的发明，如下述定义多个术语。In order to facilitate understanding of the invention disclosed below, a number of terms are defined as follows.

术语“创伤(wound)”泛指通过多种方式(例如长时间卧床休息造成的褥疮，外伤导致的创伤，切口，溃疡，烧伤等)中的任意一种导致的皮肤和皮下组织的损伤，其特征各不相同。创伤根据创伤的深度典型地可分为四个等级中的一种：(i)I级：创伤只限于上皮；(ii)II级：创伤延伸到真皮；(iii)III级：创伤延伸到皮下组织；(iv)IV级(或全厚度创伤)：暴露出骨头的创伤(例如骨头突出的压力点(bony pressure point)例如较大的转节或骶骨)。The term "wound" generally refers to damage to the skin and subcutaneous tissue caused by any of a variety of methods (such as decubitus ulcers caused by prolonged bed rest, wounds caused by trauma, incisions, ulcers, burns, etc.), which Characteristics vary. Wounds are typically classified into one of four grades according to the depth of the wound: (i) Grade I: wound limited to the epithelium; (ii) Grade II: wound extending to the dermis; (iii) Grade III: wound extending to the subcutaneous Tissue; (iv) Grade IV (or full-thickness trauma): trauma that exposes bone (eg, bony pressure point with bony prominence such as the greater trochanter or sacrum).

术语“部分厚度创伤”指包括I-III级的创伤；部分厚度创伤的实例包括烧伤，褥疮，静脉淤滞溃疡，和糖尿病溃疡。The term "partial thickness wound" is meant to include wounds of grades I-III; examples of partial thickness wounds include burns, decubitus ulcers, venous stasis ulcers, and diabetic ulcers.

术语“深度创伤”包括III级和IV级创伤。The term "deep trauma" includes grade III and grade IV wounds.

术语创伤“愈合”指例如通过形成疤痕修复创伤的过程。The term "healing" of a wound refers to the process of repairing a wound, eg, by forming a scar.

短语“诱导或加速皮肤创伤的愈合过程”指诱导创伤收缩的肉芽组织形成和／或诱导上皮形成(也就是在上皮生成新细胞)。创伤愈合可方便地通过减小的创伤面积检测。The phrase "inducing or accelerating the healing process of skin wounds" refers to inducing the formation of granulation tissue that shrinks the wound and/or inducing epithelialization (ie, the generation of new cells in the epithelium). Wound healing is conveniently measured by reduced wound area.

本发明考虑了治疗所有的创伤类型，包括深度创伤和慢性创伤。The present invention contemplates the treatment of all wound types, including deep wounds and chronic wounds.

术语“慢性创伤”指在三十天内没有愈合的创伤。The term "chronic wound" refers to a wound that does not heal within thirty days.

短语“转化细胞”指通过引入外源核酸暂时或永久地改变细胞的核酸，所述外源核酸整合到细胞基因组中，从遗传上改变所述细胞，或者保持未整合状态。The phrase "transformed cell" refers to the temporary or permanent alteration of a cell's nucleic acid by the introduction of exogenous nucleic acid that integrates into the genome of the cell, genetically alters the cell, or remains unintegrated.

本文所用的术语“顺式作用元件”是指作为DNA结合蛋白的附着位点的基因区域(例如增强子，操纵子(operator)和启动子)，从而影响同一个染色体上一个或多个基因的活性。As used herein, the term "cis-acting element" refers to a region of a gene (such as enhancers, operators, and promoters) that serves as an attachment site for DNA-binding proteins, thereby affecting the expression of one or more genes on the same chromosome. active.

本文所用的短语“反式作用因子”是指与顺式作用元件结合并调节其有关基因表达的活性的因子。例如，PDX1是与胰岛素基因启动子结合并调节其活性的反式作用因子。As used herein, the phrase "trans-acting factor" refers to a factor that binds to a cis-acting element and regulates its activity in relation to gene expression. For example, PDX1 is a trans-acting factor that binds to the insulin gene promoter and regulates its activity.

本文所用的短语“转录活化剂”是指能增加基因表达的因子。反式作用因子是直接转录活化剂的实例。As used herein, the phrase "transcriptional activator" refers to a factor that increases gene expression. Trans-acting factors are examples of direct transcriptional activators.

本文所用的术语“活化剂”是指增强活性的分子。As used herein, the term "activator" refers to a molecule that enhances activity.

本文所用的短语“被调节的表达和／或活化”是指增强的或抑制的表达和／或活化。As used herein, the phrase "modulated expression and/or activation" refers to enhanced or inhibited expression and/or activation.

PKC是一条主要的信号传导途径，其可以调节角质化细胞的增殖和分化。PKC同种型α，δ，ε，η和ζ在皮肤中表达(4，10)。在构思本发明时，设想PKC表达和／或活化的调节可以诱导细胞增殖和／或细胞分化，从而加速创伤的愈合过程。当实施本发明的时候大量实验证明了这个理论，表明PKC表达和／或活化的调节确实诱导细胞增殖和细胞分化并加速创伤的愈合过程。在进一步的具体描述中，采用了多种不同的方法调节PKC的表达和／或活化，从而加速了创伤的愈合过程。基于这些实验发现，设计了其它方法。当实施本发明的时候发现了一个引人注目的新现象-胰岛素可作为PKC表达和／或活化的调节剂。因此，胰岛素可作为调节PKC表达和／或活化的治疗剂以加速创伤的愈合过程。PKC is a major signaling pathway that regulates the proliferation and differentiation of keratinocytes. PKC isoforms α, δ, ε, η and ζ are expressed in the skin (4, 10). In conceiving the present invention, it was envisaged that modulation of PKC expression and/or activation could induce cell proliferation and/or cell differentiation, thereby accelerating the wound healing process. Extensive experiments have supported this theory when practicing the present invention, showing that modulation of PKC expression and/or activation indeed induces cell proliferation and cell differentiation and accelerates the wound healing process. In a further specific description, various methods are used to regulate the expression and/or activation of PKC, thereby accelerating the wound healing process. Based on these experimental findings, additional methods were devised. A striking new phenomenon was discovered when practicing the present invention - insulin can act as a modulator of PKC expression and/or activation. Therefore, insulin can be used as a therapeutic agent to regulate PKC expression and/or activation to accelerate the wound healing process.

不同的PKC同种型的特征以及它们对于细胞增殖和／或分化的效果对于皮肤创伤愈合生物学是非常重要的。使用PKC腺病毒构建体能够鉴别各种PKC同种型对于体外和体内创伤愈合过程的特定作用。所有同种型都能够特定地影响角质化细胞生长和分化的不同方面。两个同种型，PKCδ和PKCζ能够特定地调节整联蛋白的调控(参见下述实施例6)，与基底膜的附着(参见下述实施例9)和半桥粒的形成(参见下述实施例8)。发现两种同种型，PKCδ和PKCη调节表皮角质化细胞的增殖潜力(参见下述实施例7和11)。另外，PKCη的显性失活同种型(DNPKCη)特别能够在积极增殖的角质化细胞中诱导分化(参见下述实施例12)。最后，还在体内系统中证实了不同PKC同种型对于皮肤创伤愈合过程的作用。使用其中不同PKC同种型的表达被破坏的PKC无效的小鼠，本文显示了皮肤角质化细胞的附着和迁移过程所必需的PKCδ和PKCζ对于动物模型中体内创伤愈合过程也是很重要的。(参见实施例19)。PKC无效的皮肤的整个皮肤的全厚度活组织检查表明PKCδ和PKCζ而不是PKCα对于创伤的正确愈合是必需的。而且，下述实施例22显示了PKCα抑制剂有效促进体内创伤愈合，因而表明PKCα同种型可能对创伤愈合具有拮抗作用。The characterization of the different PKC isoforms and their effect on cell proliferation and/or differentiation is of great importance to skin wound healing biology. The use of PKC adenoviral constructs enables the identification of specific roles of various PKC isoforms on the wound healing process in vitro and in vivo. All isoforms are capable of specifically affecting different aspects of keratinocyte growth and differentiation. Two isoforms, PKCδ and PKCζ, are capable of specifically regulating integrin regulation (see Example 6 below), attachment to basement membranes (see Example 9 below) and hemidesmosome formation (see Example 9 below). Example 8). Two isoforms, PKCδ and PKCη, were found to regulate the proliferative potential of epidermal keratinocytes (see Examples 7 and 11 below). In addition, the dominant negative isoform of PKCn (DNPKCn) is particularly capable of inducing differentiation in actively proliferating keratinocytes (see Example 12 below). Finally, the role of different PKC isoforms on the skin wound healing process was also demonstrated in an in vivo system. Using PKC-null mice in which the expression of different PKC isoforms is disrupted, it is shown herein that PKCδ and PKCζ, which are essential for the attachment and migration process of dermal keratinocytes, are also important for the in vivo wound healing process in animal models. (See Example 19). Full-thickness biopsies of whole skin from PKC-null skin indicated that PKCδ and PKCζ, but not PKCα, were essential for proper wound healing. Furthermore, Example 22 below shows that PKCa inhibitors are effective in promoting wound healing in vivo, thus suggesting that PKCa isoforms may be antagonistic to wound healing.

PKCη具有独特的组织分布。它主要在上皮组织中表达(27，28)。原位杂交研究和免疫组织化学研究证明PKCη在正在分化的和分化的(differentiating and differentiative)层中高度表达(27)。本文的结果表明根据细胞生理学PKCη可作为皮肤增殖和分化的功能性调节剂。当角质化细胞在低Ca²⁺条件下维持在增殖状态的时候，PKCη诱导增殖的速度为对照角质化细胞的五到七倍。但是，当通过增加Ca²⁺浓度诱导细胞分化的时候，与对照细胞相比分化以更快和更高的速度被诱导(参见实施例12)。这可以解释PKCη显著诱导创伤愈合以及肉芽组织形成的能力，因为增殖能力和分化层形成都实现了。有趣的是，体内创伤愈合结果和PKCη在胚胎组织中的表达，其通常在成年期不以高水平表达PKCη，表明PKCη在其它组织的增殖和组织结构中可能具有作用。这包括神经以及真皮和肌肉组织，它们可在创伤的肉芽组织中有效愈合。而且，通过使用显性失活(dominant negative)突变体在积极增殖的细胞中特别调节角质化细胞的分化和诱导正常分化的能力使得特别可以操纵分化并控制创伤愈合过程中的过度增殖失调。PKCη has a unique tissue distribution. It is mainly expressed in epithelial tissues (27, 28). In situ hybridization studies and immunohistochemical studies demonstrated that PKCn is highly expressed in differentiating and differentiating layers (27). The results herein suggest that PKCn may act as a functional regulator of skin proliferation and differentiation in terms of cell physiology. When keratinocytes were maintained in a proliferative state under low Ca²⁺ conditions, PKCη-induced proliferation was five to seven times that of control keratinocytes. However, when cells were induced to differentiate by increasing the Ca2⁺ concentration, differentiation was induced at a faster and higher rate compared to control cells (see Example 12). This may explain the ability of PKCη to significantly induce wound healing as well as granulation tissue formation, as both proliferative capacity and differentiation layer formation are achieved. Interestingly, the in vivo wound healing results and the expression of PKCn in embryonic tissues, which do not normally express PKCn at high levels in adulthood, suggest a possible role for PKCn in the proliferation and organization of other tissues. This includes nerves as well as dermal and muscular tissues, which heal effectively in the granulation tissue of the wound. Furthermore, the ability to specifically regulate keratinocyte differentiation and induce normal differentiation in actively proliferating cells through the use of dominant negative mutants makes it possible in particular to manipulate differentiation and control hyperproliferative dysregulation during wound healing.

本文举例说明了在裸鼠背部产生的创伤上，PKCη的愈合能力是体内施加的。下述实施例14表明在局部感染后四天，对创伤施用PKCη表达构建体导致肉芽组织的形成。This paper exemplifies that the healing ability of PKCn is exerted in vivo on wounds produced on the back of nude mice. Example 14 below demonstrates that administration of a PKCn expression construct to a wound results in the formation of granulation tissue four days after topical infection.

总的来说，本文的结果证明了调节不同PKC同种型的表达和／或活化(膜迁移)是对抗创伤的有效工具。相应地，可以通过增加同种型PKCδ，PKCη和PKCζ的表达和／或活性，或者通过抑制同种型PKCα的表达和／或活性促进创伤愈合。Taken together, the results herein demonstrate that modulating the expression and/or activation (membrane migration) of different PKC isoforms is an effective tool in combating wounding. Accordingly, wound healing can be promoted by increasing the expression and/or activity of the isoforms PKCδ, PKCη and PKCζ, or by inhibiting the expression and/or activity of the isoform PKCα.

因此，根据本发明的一个方面，提供了一种诱导或加速皮肤创伤或损伤的愈合过程的方法，所述方法通过给皮肤创伤施用治疗有效量的至少一种调节PKC表达和／或活化的试剂来实现。根据本发明的该方面，用于实施该方法的药物组合物因此包括作为活性成分的治疗有效量的至少一种用于调节PKC表达和／或活化的试剂；和药学可接受的载体。Therefore, according to one aspect of the present invention, there is provided a method of inducing or accelerating the healing process of a skin wound or injury by administering to the skin wound a therapeutically effective amount of at least one agent that modulates PKC expression and/or activation to fulfill. According to this aspect of the invention, the pharmaceutical composition for practicing the method thus comprises, as an active ingredient, a therapeutically effective amount of at least one agent for modulating PKC expression and/or activation; and a pharmaceutically acceptable carrier.

本文所用的短语“皮肤创伤”是指任何类型的上皮创伤，包括但不限于溃疡，例如糖尿病性溃疡，褥疮，静脉溃疡，胃溃疡和HIV相关溃疡，糖尿病相关的创伤，烧伤，晒伤，老化皮肤创伤，角膜溃疡创伤，炎性胃肠道疾病创伤，肠炎性疾病创伤，克罗恩氏病创伤，溃疡性结肠炎，痔疮，大疱性表面松解症创伤，皮肤起疱创伤，牛皮癣创伤，动物皮肤创伤，动物糖尿病创伤，视网膜病创伤，口腔创伤(粘膜炎)，阴道粘膜炎创伤，牙龈病创伤，裂伤，手术切口创伤和手术后粘连创伤(adhesis wound)。The phrase "cutaneous wound" as used herein refers to any type of epithelial wound including, but not limited to, ulcers such as diabetic ulcers, decubitus ulcers, venous ulcers, gastric ulcers and HIV-associated ulcers, diabetes-associated wounds, burns, sunburn, aging Skin Wounds, Corneal Ulcer Wounds, Inflammatory Gastrointestinal Disease Wounds, Inflammatory Bowel Disease Wounds, Crohn's Disease Wounds, Ulcerative Colitis, Hemorrhoids, Bullous Surface Wounds, Skin Blistering Wounds, Psoriasis Wounds , animal skin wounds, animal diabetic wounds, retinopathy wounds, oral cavity wounds (mucositis), vaginal mucositis wounds, gingival disease wounds, lacerations, surgical incision wounds and postoperative adhesion wounds (adhesis wound).

本文所用的短语“皮肤损伤”是指任何类型的皮肤损伤或状况例如，皱纹(例如紫外线照射导致的皱纹)，皮肤纹，裂缝，肿块，大毛孔(例如与附件结构如汗腺管，皮脂腺，或毛囊相关的)，或不平或粗糙，皮肤失去弹性(功能性皮肤弹性蛋白丧失和／或失活)，下垂(包括眼部和下颌浮肿)，皮肤硬度(firmness)丧失，皮肤紧实度丧失，皮肤变形后的回复能力丧失，变色(包括黑眼圈)，斑疱，肤色灰黄，色素过量皮肤区域例如老年斑和雀斑，角质物，异常的分化，过度角质化，弹性组织变性，胶原蛋白的破坏，以及皮肤角质，真皮，表皮层，皮肤血管系统(例如毛细血管扩张或多叉血管)，以及皮下组织，特别是靠近皮肤的皮下组织中的其它组织变化。The phrase "skin damage" as used herein refers to any type of skin damage or condition, for example, wrinkles (such as those caused by ultraviolet radiation), skin lines, cracks, bumps, large pores (such as those associated with adnexal structures such as sweat ducts, sebaceous glands, or hair follicle-related), or uneven or rough, loss of skin elasticity (loss and/or inactivation of functional skin elastin), sagging (including puffiness around the eyes and jaw), loss of skin firmness, loss of skin firmness, Loss of ability to recover after skin deformation, discoloration (including dark circles), pimples, sallow complexion, hyperpigmented skin areas such as age spots and freckles, horny growths, abnormal differentiation, hyperkeratosis, elastosis, collagen destruction , and other tissue changes in the stratum corneum, dermis, epidermis, skin vasculature (such as telangiectasia or multifurcation vessels), and subcutaneous tissue, especially in the subcutaneous tissue close to the skin.

皮肤不被认为是典型的胰岛素反应性组织。因此胰岛素在皮肤中的效果主要归于它激活紧密相关的IGFR的能力。已表明在角质化细胞中，胰岛素和IGF1可以刺激受体，激活类似的下游效应子(34)。但是，本发明证明了虽然这两个生长因子都能以剂量依赖的方式诱导角质化细胞增殖，但是每种激素都是以不同的信号传导途径发挥作用。最初指出的胰岛素和IGF1对角质化细胞增殖的不同的调节由以下发现证实：这些激素以每种激素的最大增殖诱导浓度一起加入的时候对于角质化细胞增殖具有加和作用(参见实施例15)。为了鉴别胰岛素和IGF1信号传导途径在角质化细胞增殖的调控上的不同点，检测了已知的可以调节角质化细胞增殖并且作为胰岛素信号传导下游效应子的元件。这些研究表明在角质化细胞的增殖中胰岛素信号传导特别受到PKCδ的调节(参见实施例17)。在PKC蛋白家族中PKCδ是一种独特的同种型，特别参与各种细胞类型的生长和成熟(35)。但是，虽然PKCδ被证明特别受到几种生长因子包括EGF，血小板衍生生长因子和神经递质的刺激的调节，但是它的生理学作用已表明是参与了细胞生长包括细胞凋亡、分化和细胞循环延迟或停止的生长因子抑制(36-41)。近来表明在培养的小鼠角质化细胞中在升高Ca²⁺浓度之后12-24小时内，α6β4整联蛋白复合体的选择性丧失与K1的诱导互相关联(6)。α6β4蛋白表达的丧失是转录和翻译后事件，包括对α6和β4链的处理的增加，导致的。在初步的研究中在PKC的活化和α6β4整联蛋白的处理和调节之间建立起了联系。这些结果与有关PKCδ以及PKCζ对于α6β4表达的丧失和诱导角质化细胞的分离的半桥粒形成的作用的以前的结果是一致的。然而，本发明发现了PKCδ的另一个作用，就是作为胰岛素诱导的角质化细胞增殖的靶。下述实施例表明只有胰岛素的刺激，而不是多种生长因子，包括但不限于EGF，KGF，PDGF，ECGF和IGF1，能够使PKCδ，而不是皮肤中表达的任何其它PKC同种型迁移并使其活化。当EGF，KGF，PDGF，ECGF和IGF1的促有丝分裂刺激没有被PKCδ的显性失活突变体阻断的时候，PKCδ对胰岛素刺激的重要性进一步得到证实，在角质化细胞增殖的调节中胰岛素似乎是这种PKC同种型的主要活化剂(参见实施例17)。但是，当角质化细胞被WT PKCδ角质化细胞感染的时候，EGF和KGF的促有丝分裂刺激增加。这表明PKCδ的活化对于通过上游信号传导途径对其它生长因子的增殖性刺激也是必需的。而且，鉴定了下游元件，其在胰岛素诱导的PKCδ活化和角质化细胞增殖中起调节作用，而且鉴定了STAT3，一种参与此过程的转录活化剂。STAT(信号传导和转录激活子)蛋白是一个转录因子家族，包括多种细胞因子和生长因子。在七个已知的STAT家族成员中，STAT3是独特的。靶定破坏STAT3而不是其它STAT家族成员导致早期胚胎致死。特别地，当在皮肤中有条件地去除STAT3的时候，皮肤的重建被严重破坏。一旦被激活，STAT蛋白形成同源二聚体或异源二聚体，迁移到细胞核中，与靶基因的DNA反应元件结合以诱导转录。发现在角质化细胞中，PKCδ而不是皮肤中表达的其它PKC同种型(PKCα，ζ，η和ε)与STAT3组成型相关(参见实施例18)。而且，胰岛素通过对PKCδ的特异性激活调节STAT3的磷酸化，活化和核迁移。通过药物抑制剂，卡马拉素或者过表达显性失活PKCδ突变体导致的对PKCδ活性的抑制会阻断胰岛素诱导的STAT3活化和核移位。最终，显性失活PKCδ突变体的过表达会抑制STAT3的过表达诱导的角质化细胞增殖(参见实施例18)。这些结果表明在皮肤角质化细胞增殖中胰岛素诱导的PKCδ活性对于STAT3的转录激活的作用。由于STAT3对于皮肤重建非常重要，是多种细胞因子和生长因子的下游效应子，全部这些结果表明PKCδ的活化是通过多种皮肤生长因子介导角质化细胞增殖的主要下游元件。特别地，PKCδ是糖尿病病人的缺陷性创伤愈合的发病机理的主要候选物。PKCδ和创伤愈合之间的联系也在体内得到了确证。使用新构建的PKCδ无效小鼠，本文表明了缺少PKCδ会延迟小鼠皮肤的创伤愈合(参见实施例19)。在几种其它系统中也确定了PKCδ和胰岛素信号传导之间的联系。例如，最近表明在肌肉培养物中，PKCδ调节胰岛素诱导的葡萄糖转运(42，43)。类似地，在过表达胰岛素受体的细胞中，表明胰岛素刺激与PKCδ的活化有关(44-46)。但是，虽然在这些研究中胰岛素介导的PKCδ的活化与胰岛素的代谢效果有关，这是第一个将PKCδ与胰岛素介导的细胞增殖联系起来的报道。鉴别了PKCδ在角质化细胞的增殖和早期分化阶段的控制中起的双重作用，其中在所述早期分化阶段中，细胞丧失了与下面的基底膜之间的附着。这表明胰岛素诱导的PKCδ作为调节皮肤的增殖与分化之间的生理平衡的主要候选者。Skin is not considered a typical insulin-responsive tissue. The effect of insulin in the skin is thus mainly due to its ability to activate the closely related IGFR. Insulin and IGF1 have been shown to stimulate receptors in keratinocytes, activating similar downstream effectors (34). However, the present invention demonstrates that although both growth factors induce keratinocyte proliferation in a dose-dependent manner, each hormone acts through a different signaling pathway. The initially noted differential regulation of keratinocyte proliferation by insulin and IGF1 was confirmed by the finding that these hormones had additive effects on keratinocyte proliferation when added together at the maximal proliferation-inducing concentrations of each hormone (see Example 15) . To identify distinct points of insulin and IGF1 signaling pathways in the regulation of keratinocyte proliferation, elements known to regulate keratinocyte proliferation and to act as downstream effectors of insulin signaling were examined. These studies indicate that insulin signaling is specifically regulated by PKCδ in the proliferation of keratinocytes (see Example 17). PKCδ is a unique isoform in the PKC protein family that is particularly involved in the growth and maturation of various cell types (35). However, although PKCδ has been shown to be specifically regulated by stimulation by several growth factors including EGF, platelet-derived growth factor, and neurotransmitters, its physiological role has been shown to be involved in cell growth including apoptosis, differentiation, and cell cycle delay or discontinued growth factor inhibition (36-41). It was recently shown that selective loss of the α6β4 integrin complex correlates with the induction of K1 within 12-24 hours after elevated Ca2⁺ concentrations in cultured mouse keratinocytes (6). Loss of α6β4 protein expression results from transcriptional and post-translational events, including increased processing of α6 and β4 chains. A link between the activation of PKC and the processing and regulation of α6β4 integrins was established in preliminary studies. These results are consistent with previous results on the role of PKCδ and PKCζ on loss of α6β4 expression and induction of isolated hemidesmosome formation in keratinocytes. However, the present inventors discovered another role for PKCdelta as a target for insulin-induced keratinocyte proliferation. The following examples demonstrate that stimulation of insulin alone, but not multiple growth factors, including but not limited to EGF, KGF, PDGF, ECGF, and IGF1, is capable of mobilizing PKCδ, but not any other PKC isoforms expressed in the skin, and causing its activation. The importance of PKCδ for insulin stimulation was further confirmed when the mitogenic stimulation of EGF, KGF, PDGF, ECGF and IGF1 was not blocked by dominant negative mutants of PKCδ, and insulin appeared to be involved in the regulation of keratinocyte proliferation. is the major activator of this PKC isoform (see Example 17). However, the mitogenic stimulation of EGF and KGF increased when keratinocytes were infected with WT PKCδ keratinocytes. This suggests that activation of PKCδ is also necessary for proliferative stimulation of other growth factors through upstream signaling pathways. Furthermore, downstream elements were identified that play regulatory roles in insulin-induced PKCδ activation and keratinocyte proliferation, and STAT3, a transcriptional activator involved in this process, was identified. STAT (Signal Transducers and Activators of Transcription) proteins are a family of transcription factors that include a variety of cytokines and growth factors. Among the seven known STAT family members, STAT3 is unique. Targeted disruption of STAT3 but not other STAT family members results in early embryonic lethality. In particular, when STAT3 was conditionally removed in the skin, skin remodeling was severely disrupted. Once activated, STAT proteins form homodimers or heterodimers, migrate into the nucleus, and bind to DNA response elements of target genes to induce transcription. It was found that in keratinocytes, PKCδ, but not the other PKC isoforms expressed in skin (PKCα, ζ, η and ε), were constitutively associated with STAT3 (see Example 18). Furthermore, insulin regulates STAT3 phosphorylation, activation and nuclear translocation through specific activation of PKCδ. Inhibition of PKCδ activity by pharmacologic inhibitors, kamalacin, or overexpression of a dominant-negative PKCδ mutant blocked insulin-induced STAT3 activation and nuclear translocation. Finally, overexpression of a dominant negative PKCδ mutant inhibited keratinocyte proliferation induced by overexpression of STAT3 (see Example 18). These results suggest a role for insulin-induced PKCδ activity for transcriptional activation of STAT3 in cutaneous keratinocyte proliferation. Since STAT3 is very important for skin remodeling and is a downstream effector of various cytokines and growth factors, together these results suggest that the activation of PKCδ is a major downstream element in mediating keratinocyte proliferation by various skin growth factors. In particular, PKCδ is a prime candidate for the pathogenesis of defective wound healing in diabetic patients. The link between PKCδ and wound healing was also confirmed in vivo. Using newly constructed PKC[delta]-null mice, it is shown herein that the absence of PKC[delta] delays wound healing in mouse skin (see Example 19). The link between PKCδ and insulin signaling has also been identified in several other systems. For example, PKCδ was recently shown to regulate insulin-induced glucose transport in muscle cultures (42, 43). Similarly, insulin stimulation has been shown to be associated with activation of PKCδ in cells overexpressing the insulin receptor (44-46). However, while insulin-mediated activation of PKCδ was associated with the metabolic effects of insulin in these studies, this is the first report linking PKCδ to insulin-mediated cell proliferation. A dual role for PKCδ was identified in the proliferation of keratinocytes and in the control of early differentiation stages in which cells lose their attachment to the underlying basement membrane. This suggests that insulin-induced PKCδ serves as a prime candidate for regulating the physiological balance between proliferation and differentiation of the skin.

因此，根据本发明的教导，通过使创伤细胞接触胰岛素来调节PKC表达和／或活化。这可以通过多种方式实现，如下述的进一步例示。Thus, in accordance with the teachings of the present invention, PKC expression and/or activation is modulated by exposing wounded cells to insulin. This can be achieved in a number of ways, as further exemplified below.

一种方式是直接将胰岛素给予创伤。如下述实施例21和22所述，以0.1-10μM的浓度对创伤局部施用胰岛素有效促进表皮和真皮闭合以及随后的创伤愈合。然而，令人惊讶的和出乎意料的是，胰岛素和PDGF-BB生长因子，或者和PKCα抑制剂组合施用比单独施用胰岛素对创伤愈合过程产生了显著的和协同性的促进。One way is to administer insulin directly to the wound. As described in Examples 21 and 22 below, topical administration of insulin to wounds at concentrations of 0.1-10 [mu]M was effective in promoting epidermal and dermal closure and subsequent wound healing. However, surprisingly and unexpectedly, administration of insulin in combination with PDGF-BB growth factor, or with a PKCα inhibitor, resulted in a significant and synergistic enhancement of the wound healing process compared to insulin alone.

因此，根据本发明的另一个方面，提供了一种诱导或加速皮肤创伤或损伤的愈合过程的方法。所述方法通过给皮肤创伤施用治疗有效量的胰岛素和至少一种与胰岛素协同作用的其它试剂来实现，以诱导或加速皮肤创伤或损伤的愈合过程。优选地，所述试剂是PKCα抑制剂。进一步优选地，所述试剂是生长因子例如PDGF，EGF，TGFβ，KGF，ECGF或IGF1，最优选地所述试剂是PDGF-BB。Thus, according to another aspect of the present invention there is provided a method of inducing or accelerating the healing process of a skin wound or injury. The method is accomplished by administering to a skin wound a therapeutically effective amount of insulin and at least one other agent that acts synergistically with the insulin to induce or accelerate the healing process of the skin wound or injury. Preferably, the agent is a PKCα inhibitor. Further preferably, the agent is a growth factor such as PDGF, EGF, TGFβ, KGF, ECGF or IGF1, most preferably the agent is PDGF-BB.

胰岛素的直接给药，或者单独给药或者与另一种试剂组合给药，可以通过单次或重复应用来实现。在实施本发明的时候，发明人惊讶地发现以1μM的浓度单次施用胰岛素对于创伤愈合比以类似浓度进行七次的每天重复给药显著更有效(参见下述实施例20)。Direct administration of insulin, either alone or in combination with another agent, can be accomplished by single or repeated applications. In practicing the present invention, the inventors surprisingly found that a single administration of insulin at a concentration of 1 μΜ was significantly more effective for wound healing than seven repeated daily administrations at a similar concentration (see Example 20 below).

因此，根据本发明的另一个方面，提供了一种诱导或加速皮肤创伤或损伤的愈合过程的方法，包括给皮肤创伤施用单一剂量单位的治疗有效量的胰岛素。优选所述单一剂量单位包括0.001到5nM，优选0.01到0.5nM胰岛素的，例如，水溶液，凝胶，霜剂，糊剂，洗剂，喷雾，悬液，粉末，分散剂，油膏剂或软膏剂制剂，其量足以覆盖1cm面积的皮肤创伤，例如0.01-0.2ml。Thus, according to another aspect of the present invention there is provided a method of inducing or accelerating the healing process of a skin wound or injury comprising administering to the skin wound a single dosage unit of a therapeutically effective amount of insulin. Preferably said single dosage unit comprises 0.001 to 5 nM, preferably 0.01 to 0.5 nM insulin, e.g., aqueous solution, gel, cream, paste, lotion, spray, suspension, powder, dispersion, ointment or ointment Formulation in an amount sufficient to cover a skin wound of 1 cm area, for example 0.01-0.2 ml.

将胰岛素施用在创伤上的时间安排可能很重要，如下述实施例部分中的实施例20中所述。例如对4天的创伤单次施用胰岛素产生有效的创伤愈合。因此，根据本发明的另一个方面，提供了一种诱导或加速陈旧皮肤创伤的愈合过程的方法，包括给皮肤创伤施用单一剂量的治疗有效量的胰岛素。The timing of insulin administration to the wound may be important, as described in Example 20 in the Examples section below. For example a single administration of insulin to a 4 day wound produces effective wound healing. Thus, according to another aspect of the present invention there is provided a method of inducing or accelerating the healing process of an old skin wound comprising administering to the skin wound a single dose of a therapeutically effective amount of insulin.

本文所用的短语“陈旧皮肤创伤”是指至少一天的，至少两天的，至少三天的，优选至少四天的皮肤创伤。The phrase "old skin wound" as used herein refers to a skin wound that is at least one day old, at least two days old, at least three days old, preferably at least four days old.

根据本发明的另一个方面，用于诱导或加速皮肤创伤或损伤的愈合过程的药物组合物包括，作为活性成分的，治疗有效量的胰岛素，至少一种与胰岛素协同作用的其它试剂，以及设计用于药物组合物的局部应用的药学可接受的载体。优选地，所述试剂是PKCα抑制剂或生长因子例如PDGF，EGF，TGFβ，KGF，ECGF或IGF1，最优选是PDGF-BB。药学可接受的载体可以是但不限于凝胶，霜剂(cream)，糊剂，洗剂，喷雾，悬液，粉末，分散剂，油膏剂(salve)和软膏剂，下文将进一步详细描述。还可以使用固体支持物以对创伤延长释放胰岛素。应当理解所述胰岛素可以是天然的或者优选是重组的，可以是人源的或是任何其它合适来源的。According to another aspect of the present invention, a pharmaceutical composition for inducing or accelerating the healing process of skin wounds or injuries comprises, as an active ingredient, a therapeutically effective amount of insulin, at least one other agent that acts synergistically with insulin, and designed A pharmaceutically acceptable carrier for topical application of the pharmaceutical composition. Preferably, the agent is a PKCα inhibitor or a growth factor such as PDGF, EGF, TGFβ, KGF, ECGF or IGF1, most preferably PDGF-BB. Pharmaceutically acceptable carriers can be, but are not limited to, gels, creams, pastes, lotions, sprays, suspensions, powders, dispersions, salves and ointments, as described in further detail below. Solid supports can also be used for extended release of insulin to the wound. It will be appreciated that said insulin may be native or preferably recombinant, of human origin or of any other suitable origin.

根据本发明的另一个方面，用于诱导或加速皮肤创伤或损伤的愈合过程的药物组合物可以包括所选择的能够诱导或加速皮肤创伤或损伤的愈合过程的单一剂量单位的胰岛素，以及设计用于药物组合物的局部施用的药学可接受的载体。优选地，所述单一剂量单位的胰岛素是在0.01-0.2ml制剂的剂量单位中的0.001到5nM，优选0.01到0.5nM。According to another aspect of the present invention, the pharmaceutical composition for inducing or accelerating the healing process of skin wound or injury may comprise a single dosage unit of insulin selected to induce or accelerate the healing process of skin wound or injury, and designed for A pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition. Preferably, said single dosage unit of insulin is 0.001 to 5 nM, preferably 0.01 to 0.5 nM in a dosage unit of 0.01-0.2 ml formulation.

在本发明的另一个实施方案中，将表达和分泌胰岛素的细胞植入创伤，以诱导或加速皮肤创伤或损伤的愈合过程。这种能产生胰岛素的细胞可以是天然产生胰岛素的细胞，或者可以是被转化以产生和分泌胰岛素的细胞。所述细胞可以用例如重组PDX1基因(参见GeneBankAccession No.AH005712，AF035260，AF035259)转化，该基因是产生和分泌胰岛素的反式作用因子。或者，所述细胞可以通过基因敲入的方式用顺式作用元件序列转化，例如整合到所述细胞的内源胰岛素基因上游的强的组成型或诱导型启动子，以转化细胞以过度产生和分泌天然胰岛素。这是可以实现的，因为胰岛素基因的上游区域已经被克隆(参见Accession No.E00011，NM000207)。另外，所述细胞可以用重组胰岛素基因(例如Accession No.J02547)转化，因此使得所述细胞产生和分泌重组胰岛素。In another embodiment of the present invention, insulin-expressing and secreting cells are implanted into wounds to induce or accelerate the healing process of skin wounds or injuries. Such insulin-producing cells may be cells that naturally produce insulin, or may be cells that have been transformed to produce and secrete insulin. The cells can be transformed with, for example, the recombinant PDX1 gene (see GeneBank Accession Nos. AH005712, AF035260, AF035259), which is a trans-acting factor for the production and secretion of insulin. Alternatively, the cells can be transformed by knock-in with a cis-acting element sequence, such as a strong constitutive or inducible promoter integrated upstream of the cell's endogenous insulin gene, to transform the cells to overproduce and Secretes natural insulin. This is possible because the upstream region of the insulin gene has been cloned (see Accession No. E00011, NM000207). Alternatively, the cells can be transformed with a recombinant insulin gene (eg, Accession No. J02547), thus causing the cells to produce and secrete recombinant insulin.

因此根据本发明的该方面，用于诱导或加速皮肤创伤或损伤的愈合过程的药物组合物包括，作为活性成分的，胰岛素分泌细胞，以及设计用于药物组合物的局部施用的药学可接受的载体。有利的是，施用给创伤的胰岛素分泌细胞能形成分泌颗粒，因而分泌所产生的胰岛素。所述胰岛素分泌细胞可以是内分泌细胞。其可以是人源的或组织相容性人化的动物来源的。最优选地，所述胰岛素分泌细胞或者是转化的，或者是自体来源的。由所述胰岛素分泌细胞分泌的胰岛素优选是人胰岛素或者具有人胰岛素的氨基酸序列。所述胰岛素分泌细胞可以是成纤维细胞，上皮细胞或角质化细胞，前提是如上所述进行转化以使这些细胞产生和分泌胰岛素。Thus according to this aspect of the invention, a pharmaceutical composition for inducing or accelerating the healing process of a skin wound or injury comprises, as an active ingredient, insulin-secreting cells, and a pharmaceutically acceptable pharmaceutical composition designed for topical administration of the pharmaceutical composition. carrier. Advantageously, insulin secreting cells administered to a wound are capable of forming secretory granules, thereby secreting the produced insulin. The insulin-secreting cells may be endocrine cells. It may be of human origin or a histocompatible humanized animal origin. Most preferably, said insulin-secreting cells are either transformed or of autologous origin. The insulin secreted by said insulin-secreting cells is preferably human insulin or has the amino acid sequence of human insulin. The insulin-secreting cells may be fibroblasts, epithelial cells or keratinocytes, provided they are transformed as described above so that these cells produce and secrete insulin.

在另一个实施方案中，皮肤创伤的细胞被转化以产生和分泌胰岛素，以诱导或加速皮肤创伤的愈合过程。In another embodiment, cells of a skin wound are transformed to produce and secrete insulin to induce or accelerate the healing process of the skin wound.

因此根据本发明的该方面，用于诱导或加速皮肤创伤的愈合过程的药物组合物包括，作为活性成分的，设计用于转化皮肤创伤细胞以产生和分泌胰岛素的核酸构建体，以及设计用于药物组合物的局部施用的药学可接受的载体。Thus according to this aspect of the invention, a pharmaceutical composition for inducing or accelerating the healing process of skin wounds comprises, as an active ingredient, a nucleic acid construct designed to transform skin wound cells to produce and secrete insulin, and a nucleic acid construct designed for A pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

上述的任何一种转化方法，例如用编码胰岛素的构建体转化，用含有将通过基因敲入的方式插入到内源胰岛素基因下游的顺式作用元件的构建体转化，以及用编码反式作用因子的构建体转化以激活内源胰岛素的产生和分泌，都可以用于本发明的实施方案中。Any of the transformation methods described above, such as transformation with a construct encoding insulin, transformation with a construct containing a cis-acting element that will be inserted downstream of the endogenous insulin gene by gene knock-in, and transformation with a trans-acting factor encoding Transformation of constructs to activate endogenous insulin production and secretion can be used in embodiments of the present invention.

由于生命期短，可能发生分化以及无法分离稳定的转化子，很难用传统方法将外源基因有效引入原代细胞，从而阻碍了以前对于不同PKC同种型对于皮肤的效果的研究。为了克服这些障碍，使用病毒载体引入感兴趣的基因。对病毒基因组进行修饰得到复制缺陷型病毒形式的病毒载体。最广泛使用的病毒载体是逆转录病毒和腺病毒，它们可用于实验目的和基因治疗目的(13)。特别地，与逆转录载体相比，在不复制型细胞中腺病毒感染的高效率，病毒的高滴度和转导蛋白(transduced protein)的高表达使得该系统可非常有利地用于原代培养物。由于腺病毒不整合到宿主基因组中，稳定的病毒滴度可以成为复制缺陷型，因此该病毒构建体在人和动物模型中引起恶性肿瘤的危险最小(14)。至今为止，在皮肤中，已经成功使用腺病毒构建体在离体和体内方法中进行高效感染(15，16)。在本研究中使用由I.Saito和他的同事们开发的一种腺病毒载体(17)。粘粒盒(pAxCAwt)具有几乎是全长的腺病毒5基因组，但是不含有E1A，E1B和E3区域，使得该病毒为复制缺陷型。它含有复合的CAG启动子，由巨细胞病毒立即早期增强子，小鸡β-肌动蛋白启动子，和兔β-球蛋白多聚腺苷酸化信号组成，可强烈诱导插入的DNA的表达(13，17)。感兴趣的基因插入到粘粒盒中，然后与腺病毒DNA末端蛋白复合体(TPC)一起共转染到人胚胎肾293细胞中。在表达E1A和E1B区域的293细胞中，在粘粒盒和腺病毒DNA-TPC之间发生重组，以传统方法的一百倍效率产生所需的重组病毒。这种高效率主要是由于使用腺病毒DNA-TPC代替了蛋白质(proteinesed)DNA。而且，较长同源区域的存在增加了同源重组的效率。由于存在多个EcoT221位点，避免了复制型病毒的再生。应当注意，角质化细胞是用不同的PKC重组腺病毒感染的，证明24小时后有效过表达PKC同种型(参见实施例1)。Efficient introduction of exogenous genes into primary cells using traditional methods has been difficult due to short lifespan, potential for differentiation, and inability to isolate stable transformants, hampering previous studies of the effects of different PKC isoforms on the skin. To overcome these obstacles, viral vectors are used to introduce the gene of interest. Modifications to the viral genome result in viral vectors in the form of replication-defective viruses. The most widely used viral vectors are retroviruses and adenoviruses, which can be used for experimental purposes as well as for gene therapy purposes (13). In particular, the high efficiency of adenovirus infection in non-replicating cells compared to retroviral vectors, the high titer of the virus and the high expression of the transduced protein make this system very advantageous for use in primary Cultures. Since adenoviruses do not integrate into the host genome, stable viral titers can become replication deficient, and thus this viral construct poses minimal risk of malignancy in humans and animal models (14). To date, in the skin, adenoviral constructs have been successfully used for efficient infection in ex vivo and in vivo methods (15, 16). An adenoviral vector developed by I. Saito and colleagues was used in this study (17). The cosmid cassette (pAxCAwt) has an almost full-length adenovirus 5 genome, but does not contain the E1A, E1B and E3 regions, making the virus replication-defective. It contains a composite CAG promoter consisting of the cytomegalovirus immediate early enhancer, chicken β-actin promoter, and rabbit β-globin polyadenylation signal, which strongly induces the expression of inserted DNA ( 13, 17). The gene of interest was inserted into a cosmid cassette and then co-transfected into human embryonic kidney 293 cells together with the adenoviral DNA terminal protein complex (TPC). In 293 cells expressing the E1A and E1B regions, recombination occurred between the cosmid cassette and the adenoviral DNA-TPC, producing the desired recombinant virus with a hundred-fold efficiency of conventional methods. This high efficiency is mainly due to the use of adenoviral DNA-TPC instead of proteinesed DNA. Furthermore, the presence of longer regions of homology increases the efficiency of homologous recombination. Regeneration of replicating virus is avoided due to the presence of multiple EcoT221 sites. It should be noted that keratinocytes were infected with different PKC recombinant adenoviruses, demonstrating efficient overexpression of PKC isoforms after 24 hours (see Example 1).

因此，根据本发明，另一种调节PKC表达和／或活化的方法是通过在皮肤创伤细胞中诱导PKC过表达。这可以通过用顺式作用元件序列转化细胞来实现，所述顺式作用元件序列通过同源重组整合到细胞内源蛋白激酶C的上游从而使所述细胞产生天然蛋白激酶C。另外，这也可以通过用重组蛋白激酶C基因转化细胞实现，例如但不限于PKC-β1基因(Accession No.X06318，NM002738)，PKC-β2基因(AccessionNo.X07109)，PKC-γ基因(Accession No.L28035)，PKC-θ基因(Accession No.L07032)，PKC-λ基因(Accession No.D28577)，PKC-ι基因(Accession No.L18964)，PKC-α基因(AccessionNo.X52479)，PKC-δ基因(Accession No.L07860，L07861)，PKC-ε基因(Accession No.X72974)，PKC-η基因(Accession No.Z15108)和PKC-ζ基因(Accession No.Z15108，X72973，NM002744)，从而使所述细胞产生重组蛋白激酶C。Therefore, according to the present invention, another method of modulating PKC expression and/or activation is by inducing PKC overexpression in skin wound cells. This can be achieved by transforming the cell with a cis-acting element sequence integrated by homologous recombination upstream of the cell's endogenous protein kinase C such that the cell produces native protein kinase C. Alternatively, this can also be achieved by transforming cells with recombinant protein kinase C genes, such as but not limited to PKC-β1 gene (Accession No. X06318, NM002738), PKC-β2 gene (Accession No. X07109), PKC-γ gene (Accession No. .L28035), PKC-θ gene (Accession No.L07032), PKC-λ gene (Accession No.D28577), PKC-ι gene (Accession No.L18964), PKC-α gene (Accession No.X52479), PKC-δ Gene (Accession No.L07860, L07861), PKC-ε gene (Accession No.X72974), PKC-η gene (Accession No.Z15108) and PKC-ζ gene (Accession No.Z15108, X72973, NM002744), so that all The cells produce recombinant protein kinase C.

根据本发明的该方面，用于诱导或加速皮肤创伤的愈合过程的药物组合物包括，作为活性成分的，设计用于转化皮肤创伤细胞以产生蛋白激酶C的核酸构建体，以及设计用于所述药物组合物局部施用的药学可接受的载体。According to this aspect of the invention, the pharmaceutical composition for inducing or accelerating the healing process of skin wounds comprises, as an active ingredient, a nucleic acid construct designed to transform skin wound cells to produce protein kinase C, and a nucleic acid construct designed for the A pharmaceutically acceptable carrier for topical administration of the pharmaceutical composition.

根据本发明，另一种调节PKC表达和／或活化的方法是用PKC活化剂，例如但不限于Ca²⁺，胰岛素或苔藓虫素1来实现的，以诱导或加速皮肤创伤的愈合过程。According to the present invention, another method of regulating PKC expression and/or activation is to use PKC activators, such as but not limited to Ca²⁺ , insulin orbryostatin 1, to induce or accelerate the healing process of skin wounds.

因此根据本发明的此方面，用于诱导或加速皮肤创伤或损伤的愈合过程的药物组合物包括，作为活性成分的，治疗有效量的PKC活化剂，以诱导或加速皮肤创伤或损伤的愈合过程，以及药学可接受的载体。Thus according to this aspect of the present invention, the pharmaceutical composition for inducing or accelerating the healing process of skin wounds or injuries comprises, as an active ingredient, a therapeutically effective amount of a PKC activator to induce or accelerate the healing process of skin wounds or injuries , and a pharmaceutically acceptable carrier.

根据本发明，另一种调节PKC表达和／或活化的方法是下调PKC同种型的表达和／或活性。According to the present invention, another method of modulating PKC expression and/or activation is to down-regulate the expression and/or activity of a PKC isoform.

PKC同种型的活性的下调可以使用PKC伪底物同种型抑制剂，例如PKCα，PKCζ或PKCη伪底物抑制剂(CalbioChem，California USA)，或者另一种PKC同种型抑制剂例如药用肽LY379196(Elly Lilly，USA)。Downregulation of the activity of a PKC isoform can use a PKC pseudosubstrate isoform inhibitor, such as a PKCα, PKCζ or PKCη pseudosubstrate inhibitor (CalbioChem, California USA), or another PKC isoform inhibitor such as a drug Peptide LY379196 (Elly Lilly, USA) was used.

或者，PKC同种型的活性的下调可以用显性失活(DN)PKC腺病毒构建体来实现，如下述实施例部分所述。Alternatively, downregulation of the activity of a PKC isoform can be achieved using a dominant negative (DN) PKC adenoviral construct, as described in the Examples section below.

PKC同种型的表达的下调可以使用小干扰RNA(siRNA)分子。RNA干扰是一个两步过程。第一步，定义为起始步骤，可能是通过Dicer，dsRNA特异性核糖核酸酶的RNase III家族的成员的作用，以依赖于ATP的方式处理(切割)dsRNA(直接引入或通过转基因或病毒)，将输入的dsRNA消化为21-23个核苷酸(nt)的小干扰RNA(siRNA)。后续切割将会将RNA降解为19-21bp的双链(siRNA)，每个都有2’-核苷酸3’突出端[Hutvagner and Zamore Curr.Opin.Genetics andDevelopment12：225-232(2002)；和Bernstein Nature409：363-366(2001)]。Downregulation of expression of PKC isoforms can use small interfering RNA (siRNA) molecules. RNA interference is a two-step process. The first step, defined as the initiation step, is probably through the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) the dsRNA in an ATP-dependent manner (directly introduced or by transgenes or viruses) , digest the input dsRNA into 21-23 nucleotide (nt) small interfering RNA (siRNA). Subsequent cleavage will degrade the RNA into 19-21 bp duplexes (siRNAs), each with 2'-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].

在效应步骤中，siRNA双链与核酸酶复合体结合形成RNA诱导的沉默复合体(RISC)。RISC的激活需要siRNA双链以ATP依赖的方式解旋。然后活性RISC通过碱基对的相互作用靶定同源转录物，从siRNA的3’末端将mRNA切割成12个核苷酸的片段[Hutvagner and ZamoreCurr.Opin.Genetics and Development12：225-232(2002)；Hammond et al.(2001)Nat.Rev.Gen.2：110-119(2001)；和SharpGenes.Dev.15：485-90(2001)]。虽然切割的机制还有待阐明，研究表明每个RISC含有一个单个的siRNA和RNAase[Hutvagner andZamore Curr.Opin.Genetics and Development12：225-232(2002)]。In the effector step, the siRNA duplex binds to the nuclease complex to form the RNA-induced silencing complex (RISC). RISC activation requires unwinding of the siRNA duplex in an ATP-dependent manner. Active RISC then targets homologous transcripts through base-pair interactions, cleaving the mRNA into 12-nucleotide fragments from the 3' end of the siRNA [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12: 225-232 (2002 ); Hammond et al. (2001) Nat. Rev. Gen. 2: 110-119 (2001); and SharpGenes. Dev. 15: 485-90 (2001)]. Although the mechanism of cleavage has yet to be elucidated, studies have shown that each RISC contains a single siRNA and RNAase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].

由RNAi的显著效力，表明RNAi途径中具有扩增步骤。可以通过拷贝输入的dsRNA，这会产生更多的siRNA，或者通过复制形成的siRNA来扩增。另外或此外，可以通过RISC的多转换事件(multipleturnover events)实现扩增[Hammond et al.Nat.Rev.Gen.2：110-119(2001)，Sharp Genes.Dev.15：485-90(2001)；Hutvagnerand Zamore Curr.Opin.Genetics and Development12：225-232(2002)]。要获得更多关于RNAi的信息，参见下述综述TuschlChemBiochem.2：239-245(2001)；Cullen Nat.Immunol.3：597-599(2002)；和Brant l Biochem.Biophys.Act.1575：15-25(2002)。There is an amplification step in the RNAi pathway indicated by the remarkable potency of RNAi. Amplification can be achieved by copying the input dsRNA, which produces more siRNA, or by replicating the formed siRNA. Alternatively or additionally, amplification can be achieved by multiple turnover events of RISC [Hammond et al. Nat. Rev. Gen. 2: 110-119 (2001), Sharp Genes. Dev. ); Hutvagner and Zamore Curr. Opin. Genetics and Development 12: 225-232 (2002)]. For more information on RNAi, see the following reviews Tuschl Chem Biochem. 2: 239-245 (2001); Cullen Nat. Immunol. 3: 597-599 (2002); and Brant l Biochem. Biophys. Act. 1575: 15 -25 (2002).

可以如下所述合成适用于本发明的RNAi分子。首先，在PKC同种型mRNA序列的AUG起始密码子的下游寻找AA二核苷酸序列。每个AA和3’附近19个核苷酸的出现计录为潜在的siRNA靶位点。由于在调控蛋白结合位点中更富含不翻译区(UTR)，优选siRNA靶位点选自开放阅读框架。UTR结合蛋白和／或翻译起始复合物会干扰siRNA核酸内切酶复合物的结合[Tuschl ChemBiochem.2：239-245]。应当理解，针对不翻译区的siRNA也是有效的，由GAPDH证明，其中针对5’UTR的siRNA介导细胞GAPDH mRNA减少大约90％，完全破坏了蛋白水平(www.ambion.com／techlib／tn／91／912.html)。RNAi molecules suitable for use in the present invention can be synthesized as described below. First, the AA dinucleotide sequence was looked for downstream of the AUG start codon in the PKC isoform mRNA sequence. Occurrences of each AA and 19 nucleotides near 3' were counted as potential siRNA target sites. Since untranslated regions (UTRs) are more abundant in regulatory protein binding sites, it is preferred that siRNA target sites are selected from open reading frames. UTR binding proteins and/or translation initiation complexes interfere with the binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated that siRNA targeting untranslated regions is also effective as demonstrated by GAPDH, where siRNA targeting the 5' UTR mediated a reduction of cellular GAPDH mRNA by approximately 90%, completely disrupting protein levels (www.ambion.com/techlib/tn/ 91/912.html ).

其次，用任何序列比对软件，例如NCBI服务器上的BLAST软件(www.ncbi.nlm.nih.gov／BLAST／)将潜在的靶位点与合适的基因组数据库(例如人，小鼠，大鼠等)相比较。筛选出与其它编码序列具有显著同源性的推定的靶位点。Next, use any sequence alignment software, such as BLAST software on the NCBI server (www.ncbi.nlm.nih.gov/BLAST/) to align potential target sites with appropriate genomic databases (e.g., human, mouse, rat etc.) for comparison. Putative target sites were screened for significant homology to other coding sequences.

选择合格的靶序列作为siRNA合成的模板。优选的序列是具有低的G／C含量的那些，这是因为这种序列已证明与那些G／C含量高于55％的序列相比能更有效地介导基因沉默。优选沿着靶基因的长度选择几个靶位点进行评价。为了更好地评价所选的siRNA，优选同时使用负对照。负对照siRNA优选与所述siRNA具有同样的核苷酸成分，但是与基因组没有显著的同源性。因此，优选使用siRNA的混杂核苷酸序列，前提是它不会与任何其它基因具有任何显著的同源性。根据本发明，合适的siRNA可以是，例如，能抑制PKCα表达的siRNA，例如SEQ IDNO：1-6的任何核酸序列。Qualified target sequences are selected as templates for siRNA synthesis. Preferred sequences are those with a low G/C content, since such sequences have been shown to mediate gene silencing more efficiently than those with a G/C content above 55%. Preferably several target sites are selected for evaluation along the length of the target gene. In order to better evaluate the selected siRNA, it is preferable to use a negative control at the same time. The negative control siRNA preferably has the same nucleotide composition as the siRNA, but has no significant homology with the genome. Therefore, it is preferred to use a promiscuous nucleotide sequence of the siRNA, provided that it does not have any significant homology to any other gene. According to the present invention, a suitable siRNA can be, for example, an siRNA capable of inhibiting the expression of PKCα, such as any nucleic acid sequence of SEQ ID NO: 1-6.

另一种能下调PKC同种型的试剂是能特异性切割PKC同种型的mRNA转录物或DNA序列的DNA酶分子。DNA酶是能切割单链和双链靶序列的单链多核苷酸(Breaker，R.R.and Joyce，G.Chemistry andBiology1995；2：655；Santoro，S.W.&Joyce，G.F.Proc.Natl.Acad.Sci.USA1997；943：4262)。已提出了DNA酶的通用模型(“10-23”模型)。“10-23”DNA酶具有一个15个脱氧核糖核苷酸的催化结构域，侧翼是两个各自具有七个到九个脱氧核糖核苷酸的底物识别结构域。这种类型的DNA酶可以在嘌呤：嘧啶结合处有效切割其底物RNA(Santoro，S.W.&Joyce，G.F.Proc.Natl.Acad.Sci.USA199；DNA酶的综述参见Khachigian，LM[Curr Opin Mol Ther4：119-21(2002)])。Another agent capable of down-regulating a PKC isoform is a DNase molecule that specifically cleaves the mRNA transcript or DNA sequence of the PKC isoform. DNase is a single-stranded polynucleotide capable of cleaving single- and double-stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl. Acad. Sci. USA 1997; 943:4262). A general model for DNase has been proposed (the "10-23" model). The "10-23" DNase has a catalytic domain of 15 deoxyribonucleotides flanked by two substrate recognition domains of seven to nine deoxyribonucleotides each. This type of DNase efficiently cleaves its substrate RNA at the purine:pyrimidine junction (Santoro, S.W. & Joyce, G.F. Proc. Natl. Acad. Sci. USA 199; for a review of DNase see Khachigian, LM [Curr Opin Mol Ther 4: 119-21(2002)]).

构建和扩增合成的、基因工程的能识别单链的和双链的靶切割位点的DNA酶的例子已在Joyce等的U.S.Pat.No.6,326,174中公开。最近观察到类似设计的针对人尿激酶受体的DNA酶抑制尿激酶受体的表达，并成功抑制体内结肠癌细胞转移(Itoh et al，20002，Abstract409，Ann Meeting Am Soc Gen Ther www.asgt.org)。在另一种应用中，互补于bcr-abl致癌基因的DNA酶成功抑制白血病细胞中致癌基因的表达，在CML和ALL中减少自体骨髓移植的复发率。Examples of construction and amplification of synthetic, genetically engineered DNases that recognize single- and double-stranded target cleavage sites are disclosed in U.S. Pat. No. 6,326,174 by Joyce et al. A similarly designed DNase against the human urokinase receptor was recently observed to inhibit expression of the urokinase receptor and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt. org). In another application, DNase complementary to the bcr-abl oncogene successfully inhibited the expression of the oncogene in leukemia cells, reducing the relapse rate of autologous bone marrow transplantation in CML and ALL.

PKC同种型的下调还可以使用能特异与编码所述PKC同种型的mRNA转录物杂交的反义多核苷酸来实现。Downregulation of a PKC isoform can also be achieved using antisense polynucleotides that hybridize specifically to mRNA transcripts encoding said PKC isoform.

可用于有效下调PKC同种型的反义分子的设计要考虑反义技术的两个重要方面。第一个方面是寡核苷酸向合适细胞的细胞质中的递送，第二个方面是能以抑制其翻译的方式在细胞中特异结合指定mRNA的寡核苷酸的设计。The design of antisense molecules that can be used to efficiently downregulate PKC isoforms takes into account two important aspects of antisense technology. The first aspect is the delivery of oligonucleotides into the cytoplasm of suitable cells, and the second aspect is the design of oligonucleotides capable of specifically binding to a given mRNA in the cell in a manner that inhibits its translation.

现有技术给出了许多递送策略，可用于将寡核苷酸有效递送到多种细胞类型中[参见，例如Luft J Mol Med76：75-6(1998)；Kronenwett et al.Blood91：852-62(1998)；Rajur et al.Bioconjug Chem8：935-40(1997)；Lavigne et al.Biochem BiophysRes Commun237：566-71(1997)和Aoki et al.(1997)BiochemBiophys Res Commun231：540-5(1997)]。The prior art presents a number of delivery strategies that can be used to efficiently deliver oligonucleotides into a variety of cell types [see, e.g., Luft J Mol Med76:75-6 (1998); Kronenwett et al. Blood91:852-62 (1998); Rajur et al. Bioconjug Chem8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997) ].

此外，根据能解释靶mRNA和寡核苷酸中结构变化的能量的热力学循环，鉴别与它们的靶mRNA具有最高的预测的结合亲和力的序列的算法也是可获得的[参见，例如，Wal ton et al.Biotechnol Bioeng65：1-9(1999)]。In addition, algorithms are available that identify sequences with the highest predicted binding affinities to their target mRNAs based on thermodynamic cycles that account for the energy of structural changes in target mRNAs and oligonucleotides [see, e.g., Walton et al. al. Biotechnol Bioeng 65:1-9 (1999)].

这些算法已成功用于实现细胞中的反义方法。例如，Walton et al.开发的算法使得科学家成功设计出了兔β-球蛋白(RBG)和小鼠肿瘤坏死因子-α(TNFα)转录物的反义寡核苷酸。同一个研究小组最近报道了针对三个模型靶mRNA(人乳酸脱氢酶A和B和大鼠gp130)合理选择的寡核苷酸在细胞培养物中的反义活性，其是用证明在几乎所有情况下都有效的动态PCR方法评估的，包括用磷酸二酯和硫代磷酸酯寡核苷酸化学对两种细胞类型中的三个不同靶的检测。These algorithms have been successfully used to implement antisense methods in cells. For example, the algorithm developed by Walton et al. allowed scientists to successfully design antisense oligonucleotides for rabbit β-globulin (RBG) and mouse tumor necrosis factor-α (TNFα) transcripts. The same research group recently reported the antisense activity in cell culture of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gp130), which was demonstrated in almost Kinetic PCR methods that were valid in all cases were evaluated, including the detection of three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.

此外，还已公开了几种用体外系统设计和预测特定寡核苷酸的效率的方法(Matveeva et al.，Nature Biotechnology16：1374-1375(1998))。In addition, several methods for designing and predicting the efficiency of specific oligonucleotides using in vitro systems have been published (Matveeva et al., Nature Biotechnology 16:1374-1375 (1998)).

几个临床实验已证明反义寡核苷酸是安全性，可行性和活性。例如，适用于治疗癌症的反义寡核苷酸已经成功使用[Holmund etal.，Curr Opin Mol Ther1：372-85(1999)]，而用靶定c-myb基因，p53和Bc1-2的反义寡核苷酸治疗血液肿瘤也进入了临床实验，已显示被病人耐受[GerWitz Curr Opin Mol Ther1：297-306(1999)]。Several clinical experiments have demonstrated the safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther1:372-85 (1999)], while antisense oligonucleotides targeting c-myb, p53 and Bcl-2 Sense oligonucleotides have also entered clinical trials in the treatment of hematological malignancies and have been shown to be tolerated by patients [GerWitz Curr Opin Mol Ther1: 297-306 (1999)].

最近，已报道了在小鼠模型中反义介导的人乙酰肝素酶(heparanase)基因表达的抑制可抑制人癌细胞的胸膜扩散[Uno etal.，Cancer Res61：7855-60(2001)]。Recently, antisense-mediated inhibition of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60 (2001)] .

因此，目前的共识就是最近反义技术领域中的进展，如上所述，导致产生了高度精确的反义设计算法和多种寡核苷酸递送系统，使得普通技术人员能够设计和实施适用于下调已知序列的表达的反义方法而不需要过度的试验和错误实验。Thus, the current consensus is that recent advances in the field of antisense technology, as described above, have resulted in highly precise antisense design algorithms and a variety of oligonucleotide delivery systems, enabling those of ordinary skill to design and implement antisense agents suitable for downregulation. The antisense approach to expression of known sequences does not require undue trial and error experimentation.

另一种能下调PKC同种型的试剂是能特异切割编码PKC同种型的mRNA转录物的核糖酶(ribozyme)分子。核糖酶越来越多地用于基因表达的序列特异性抑制，通过切割编码感兴趣蛋白的mRNA[Welch etal.，Curr Opin Biotechnol.9：486-96(1998)]。能够设计用于切割任何特异靶RNA的核糖酶，这使得核糖酶成为基础研究和治疗应用中的很有价值的工具。在治疗领域，核糖酶用于在感染性疾病中靶定病毒RNA，在癌症中靶定优势的致癌基因以及在遗传疾病中靶定特异的体细胞变异[Welch et al.，Clin Diagn Virol.10：163-71(1998)]。最值得注意的是，有几种用于HIV患者的核糖酶基因治疗方案已经在进行I期实验。最近，核糖酶用于进行转基因动物研究，基因靶的验证和途径的阐明。有几种核糖酶正在进行不同阶段的临床实验。ANGIOZYME是第一个化学合成的用在人临床实验研究中的核糖酶。ANGIOZYME特异性抑制VEGF-r(血管内皮生长因子受体)，一种血管生成途径中的关键成分的形成。Ribozyme Pharmaceuticals，Inc.与其它公司证明了动物模型中抗血管生成治疗的重要性。HEPTAZYME，一种设计用于选择性破坏C型肝炎病毒(HCV)RNA的核糖酶，已发现在细胞培养物的检测中有效减少C型肝炎病毒RNA(RibozymePharmaceuticals，Incorporated-WEB主页)。Another agent capable of downregulating a PKC isoform is a ribozyme molecule that specifically cleaves mRNA transcripts encoding a PKC isoform. Ribozymes are increasingly used for sequence-specific inhibition of gene expression by cleaving mRNA encoding a protein of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The ability to design ribozymes to cleave any specific target RNA makes ribozymes valuable tools in basic research and therapeutic applications. In the therapeutic arena, ribozymes are used to target viral RNA in infectious diseases, predominate oncogenes in cancer, and specific somatic mutations in genetic diseases [Welch et al., Clin Diagn Virol.10 : 163-71 (1998)]. Most notably, several ribozyme gene therapy regimens for HIV patients are already in phase I trials. More recently, ribozymes have been used to perform transgenic animal studies, validation of gene targets and elucidation of pathways. Several ribozymes are in various stages of clinical trials. ANGIOZYME is the first chemically synthesized ribozyme used in human clinical trials. ANGIOZYME specifically inhibits the formation of VEGF-r (vascular endothelial growth factor receptor), a key component in the angiogenic pathway. Ribozyme Pharmaceuticals, Inc. and others have demonstrated the importance of anti-angiogenic therapy in animal models. HEPTAZYME, a ribozyme designed to selectively destroy hepatitis C virus (HCV) RNA, has been found to be effective in reducing HCV RNA in assays in cell culture (Ribozyme Pharmaceuticals, Incorporated-WEB home page).

优选地，用于诱导或加速皮肤创伤或损伤的愈合过程的药物组合物进一步包括至少一种选自由以下成员构成的组的其它试剂：激素，生长因子，脂肪细胞激素(adipokine)，PKCδRACK和GW9662。合适的激素包括，但不限于胰岛素。合适的生长因子可以是但不限于白介素-6(IL-6)，角质化细胞生长因子(KFG)或肿瘤坏死因子α(TNFα)。合适的脂肪细胞激素可以是但不限于adipsin或脂联素(adiponectin)。Preferably, the pharmaceutical composition for inducing or accelerating the healing process of skin wounds or injuries further comprises at least one other agent selected from the group consisting of hormones, growth factors, adipokine, PKCδRACK and GW9662 . Suitable hormones include, but are not limited to insulin. Suitable growth factors may be, but are not limited to, interleukin-6 (IL-6), keratinocyte growth factor (KFG) or tumor necrosis factor alpha (TNFα). A suitable adipocyte hormone may be, but is not limited to, adipsin or adiponectin.

在实施本发明的时候，发明人惊讶地和出乎意料地发现共聚物-1(glatiremar acetate)能够在体外和体内显著促进创伤愈合(参见下述实施例部分的实施例26)。以前已知共聚物-1是一种免疫调节剂，用于治疗多发性硬化和中枢神经系统疾病(美国专利号6,620,847，6,362,161，6,342,476，6,054,430，6,046,898，5,981,589和5,800,808；美国申请序列号10／615865，10／666857和10／014477)，现有技术没有描述或提示共聚物-1可用于加速创伤愈合过程。In carrying out the present invention, the inventors have surprisingly and unexpectedly discovered that Copolymer-1 (glatiremar acetate) is capable of significantly promoting wound healing both in vitro and in vivo (see Example 26 in the Examples section below). Copolymer-1 was previously known to be an immunomodulatory agent for the treatment of multiple sclerosis and central nervous system disorders (U.S. Patent Nos. 6,620,847, 6,362,161, 6,342,476, 6,054,430, 6,046,898, 5,981,589, and 5,800,808; U.S. Application Serial No. 10/615865 , 10/666857 and 10/014477), the prior art does not describe or suggest that copolymer-1 can be used to accelerate the wound healing process.

因此根据本发明的另一个方面提供了一种用于诱导或加速皮肤创伤或损伤的愈合过程的方法，所述方法包括给皮肤创伤或损伤施用治疗有效量的共聚物-1，优选浓度范围为1到500μg／ml。因此根据本发明的该方面用于实施该方法的药物组合物包括，作为活性成分的，治疗有效量的共聚物-1和药学可接受的载体。Therefore according to another aspect of the present invention there is provided a method for inducing or accelerating the healing process of a skin wound or injury, said method comprising applying a therapeutically effective amount of copolymer-1 to a skin wound or injury, preferably in a concentration range of 1 to 500 μg/ml. Thus the pharmaceutical composition for practicing the method according to this aspect of the invention comprises, as an active ingredient, a therapeutically effective amount of Copolymer-1 and a pharmaceutically acceptable carrier.

本发明的治疗／药学活性成分可以以其本身，或者与合适的载体和／或赋形剂混合成药物组合物施用给创伤。适用于本发明的药物组合物包括其中含有有效量的能获得预期治疗效果的活性成分的那些组合物。The therapeutically/pharmaceutically active ingredients of the present invention can be administered to wounds by themselves or mixed with suitable carriers and/or excipients to form a pharmaceutical composition. Pharmaceutical compositions suitable for use in the present invention include those containing the active ingredient in an effective amount to achieve the desired therapeutic effect.

这里所用的“药物组合物”指本文所述的一种或多种活性成分，蛋白质，化学品，核酸或细胞，或其生理学可接受的盐或药物前体，与其它化学组分例如传统药物，生理学合适的载体和赋形剂的制剂。药物组合物的目的是便于将化合物或细胞给予生物体。本发明的药物组合物可以用本领域公知的方法制备，例如通过传统的混合，溶解，制粒，包糖衣，粉碎，乳化，制成胶囊，包埋或冻干过程制备。As used herein, "pharmaceutical composition" refers to one or more active ingredients, proteins, chemicals, nucleic acids or cells described herein, or their physiologically acceptable salts or prodrugs, in combination with other chemical components such as traditional medicines , formulations with physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound or cell to an organism. The pharmaceutical composition of the present invention can be prepared by methods known in the art, such as traditional mixing, dissolving, granulating, sugar-coating, pulverizing, emulsifying, encapsulating, embedding or freeze-drying.

在下文中，短语“生理学合适的载体”和“药学可接受的载体”可互换使用，均指不会对生物体引起显著刺激以及不会破坏给药的结合物(conjugate)的生物活性和性质的载体或稀释剂。Hereinafter, the phrases "physiologically suitable carrier" and "pharmaceutically acceptable carrier" are used interchangeably, and both refer to a carrier that does not cause significant irritation to the organism and does not destroy the biological activity and properties of the administered conjugate (conjugate). carrier or diluent.

本文中术语“赋形剂”是指添加到药物组合物中以进一步方便所述活性成分的加工和给药的惰性物质。赋形剂的非限制性实例包括碳酸钙，磷酸钙，不同的糖和不同类型的淀粉，纤维素衍生物，明胶，植物油和聚乙二醇。The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate the processing and administration of the active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, different sugars and different types of starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

活性成分的配制和给药技术可见“Remington’s PharmaceuticalSciences，”Mack Publishing Co.，Eas ton，PA的最后一版，在此引入作为参考。Techniques for formulation and administration of active ingredients can be found in "Remington's Pharmaceutical Sciences," last edition of Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

尽管活性成分可经不同的途径给药，如前所述，为本发明的目的，局部给药方式是优选的，助以局部载体。局部载体通常适用于活性成分的局部给药，包括本领域公知的任何这种材料。为将所述组合物制成所需形式可选择局部载体，例如流体或非流体载体，洗剂，霜剂，糊剂，凝胶，粉末，软膏剂，溶剂，流体稀释剂，滴剂等，并且可以包括天然存在的或合成来源的物质。明显所选的载体必须不能对局部制剂的活性试剂或其它成分产生不良影响，对于局部制剂中的所有成分应是稳定的。可用于本文的合适的局部载体的实例包括水，酒精和其它无毒的有机溶剂，甘油，矿物油，硅氧烷，凡士林，羊毛脂，脂肪酸，植物油，对羟基苯甲酸酯，蜡等。本文中优选制剂是无色，无味的软膏，流体，洗液，霜和凝胶。Although the active ingredient may be administered by various routes, as previously stated, topical administration is preferred for the purposes of the present invention, aided by a topical carrier. Topical carriers are generally suitable for topical administration of the active ingredient and include any such materials known in the art. The topical carrier may be chosen to bring the composition into the desired form, such as fluid or non-fluid carriers, lotions, creams, pastes, gels, powders, ointments, solvents, fluid diluents, drops and the like, And may include materials of naturally occurring or synthetic origin. Obviously the carrier chosen must not adversely affect the active agent or other ingredients of the topical formulation and should be stable to all ingredients in the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicones, petrolatum, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Preferred formulations herein are colorless, odorless ointments, fluids, lotions, creams and gels.

软膏(ointment)是半固体制剂，典型地是以矿脂或其它石油衍生物为基质。本领域技术人员知道，待使用的特定的软膏基质能够提供最佳的活性成分递送，优选能够提供其它需要的特性以及例如软化作用等。与其它载体或媒介物一样，软膏基质应是惰性的，稳定的，无刺激性的不致敏的。如Remington：The Science and Practice ofPharmacy，19th Ed.(Easton，Pa.：Mack Publishing Co.，1995)的1399-1404页中指出的，软膏基质可分为四类：油质基质；可乳化基质；乳液基质；和水溶性基质。油质软膏基质包括，例如植物油，获自动物的脂肪，以及从石油获得的半固体碳氢化合物。可乳化软膏基质，也称为可吸收软膏基质，含少量或不含水，包括，例如羟基硬脂酸甘油硫酸酯，无水羊毛脂和亲水矿脂(petrolatum)。乳液软膏基质是油包水(W／O)或水包油(O／W)乳液，包括，例如十六烷醇，单硬脂酸甘油酯，羊毛脂和硬脂酸。优选的水溶性软膏基质是用不同分子量的聚乙二醇制备的；此外，进一步的信息参考Remington：TheScience and Practice of Pharmacy。Ointments are semisolid preparations, typically based on petrolatum or other petroleum derivatives. Those skilled in the art will appreciate that the particular ointment base to be used will provide optimum delivery of the active ingredient and preferably other desirable properties as well as e.g. softening. Like other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As pointed out in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), pages 1399-1404, ointment bases can be divided into four categories: oleaginous bases; emulsifiable bases; an emulsion base; and a water soluble base. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbable ointment bases, contain little or no water and include, for example, glyceryl hydroxystearate sulfate, anhydrous lanolin and hydrophilic petrolatum (petrolatum). Emulsion ointment bases are water-in-oil (W/O) or oil-in-water (O/W) emulsions and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared using polyethylene glycols of various molecular weights; also, see Remington: The Science and Practice of Pharmacy for further information.

洗液是用于皮肤表面并且不用摩擦的制剂，通常是流体或半流体制剂，其中固体颗粒，包括活性试剂，存在于水或酒精基质中。洗液通常是固体悬液，可含有水包油类型的流体油状乳液。洗液是本文中优选用于治疗身体大面积的制剂，这是由于更具流动性的组合物容易施用。通常洗液中的不溶物质必需磨碎。洗液典型地包括悬浮试剂以更好地分散，以及用于使所述活性试剂局部化和保持与皮肤接触的化合物，例如甲基纤维素，羧甲基纤维素钠等。A lotion is a non-abrasive, usually fluid or semi-fluid formulation in which solid particles, including the active agent, are present in a water or alcohol base, applied to the surface of the skin without rubbing. Lotions are usually solid suspensions and may contain fluid oily emulsions of the oil-in-water type. Lotions are preferred formulations herein for treating large areas of the body due to the ease of application of the more fluid compositions. Usually the insoluble material in the lotion must be ground. Lotions typically include suspending agents for better dispersion, and compounds for localizing and maintaining the active agent in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, and the like.

含有选定的活性成分的霜剂(creams)，如本领域公知的，是粘性流体或半固体的乳液，是水包油或油包水的。霜剂基质是可水洗的，含有油相，乳化剂和水相。油相，有时也称为“内部”相，通常包括矿脂和脂肪醇例如十六烷醇或十八烷醇；水相通常，但不是必需的，体积超过油相，通常含有湿润剂。霜剂中的乳化剂，在Remington，上文中有解释，通常是非离子的，阴离子的，阳离子的或两性的表面活性剂。Creams containing selected active ingredients, as are known in the art, are viscous fluid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are washable and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, sometimes called the "internal" phase, usually includes petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the water phase usually, but not necessarily, exceeds the oil phase in volume and usually contains a humectant. Emulsifiers in creams, as explained in Remington, supra, are usually nonionic, anionic, cationic or amphoteric surfactants.

凝胶制剂优选应用于头皮。在局部活性成分制剂领域工作的技术人员知道，凝胶是半固体，悬液类型的系统。单相凝胶含有基本上均匀分布于载体流体中的有机大分子，典型地是水性的，但是优选还含有酒精以及任选的，油。Gel formulations are preferably applied to the scalp. Those skilled in the art of topical active ingredient formulations know that gels are semi-solid, suspension-type systems. Single-phase gels contain organic macromolecules substantially uniformly distributed in a carrier fluid, typically aqueous, but preferably also alcohol and, optionally, oil.

核酸载体包括但不限于脂质体，包括靶定的脂质体，核酸络合剂，病毒包被等。但是也可以使用裸露核酸转化。Nucleic acid carriers include, but are not limited to, liposomes, including targeted liposomes, nucleic acid complexing agents, viral coatings, and the like. It is also possible, however, to use naked nucleic acid for transformation.

本发明的局部制剂中可以包含本领域技术人员公知的各种添加剂。例如，可以用溶剂溶解某些活性成分物质。其它任选的添加剂包括皮肤渗透增强剂，遮光剂，抗氧化剂，胶凝剂，增稠剂，和稳定剂等。Various additives known to those skilled in the art may be included in the topical formulations of the present invention. For example, solvents may be used to dissolve certain active ingredient materials. Other optional additives include skin penetration enhancers, opacifiers, antioxidants, gelling agents, thickeners, and stabilizers, among others.

如上所述的，根据本发明，用于治疗创伤的局部制剂可以含有传统用于治疗这种创伤的其它药学活性试剂或成分。包括免疫抑制剂，例如环孢霉素，抗代谢物，例如氨甲喋呤，皮质类固醇，维生素D和维生素D类似物，维生素A或其类似物，例如依曲替酯(etretinate)，焦油，煤焦油，抗痒和促角质化(keratoplastic)试剂，例如杜松油，角质层分离试剂，例如水杨酸，软化剂，润滑剂，防腐剂和消毒剂，例如杀菌剂蒽三酚(也称为蒽啉)，光敏剂，例如补骨脂素和甲氧补骨脂素和UV辐射。还可以加入其它试剂，例如抗微生物试剂，抗真菌试剂，抗生素和抗炎性试剂。还可以同时进行氧处理(高氧压)。As mentioned above, topical formulations for treating wounds according to the present invention may contain other pharmaceutically active agents or ingredients conventionally used for treating such wounds. Including immunosuppressants such as cyclosporine, antimetabolites such as methotrexate, corticosteroids, vitamin D and vitamin D analogs, vitamin A or its analogs such as etretinate, tar, coal tar, Anti-itch and keratoplastic agents, such as juniper oil, keratolytic agents, such as salicylic acid, emollients, emollients, antiseptics, and disinfectants, such as the fungicide anthracenol (also known as anthraline ), photosensitizers such as psoralen and methoxypsoralen, and UV radiation. Other agents such as antimicrobials, antifungals, antibiotics and anti-inflammatory agents may also be added. Oxygen treatment (hyperoxygen pressure) can also be carried out simultaneously.

本发明的局部组合物还可以用传统的皮肤贴片或用品递送到皮肤上，其中所述活性成分组合物包含于层状结构中，其中层状结构作为药物递送装置固定于皮肤上。在这样一种结构中，所述活性成分组合物含于一层，或“储库(reservoir)”中，其上面是表面垫层(upperbacking layer)。所述层状结构可含有单个储库，或者它可以含有多个储库。在一个实施方案中，所述储库含有聚合物基质的药学可接受的接触粘附物质，以在活性成分递送期间将该系统固定于皮肤上。合适的皮肤接触粘附物质的实例包括但不限于聚乙烯，聚硅氧烷，聚异丁烯，聚丙烯酸酯和聚氨酯等。特定的聚合物胶粘剂的选择取决于特定的活性成分，媒介物等，也就是，所述胶粘剂必须与含有活性成分的组合物中的所有组分是相容的。另外，含有活性成分的储库和皮肤接触胶粘剂应当是独立的和不同的层，胶粘剂在储库之下，在这种情况下，储库可以是如上所述的聚合物基质，或者可以是液体或水凝胶储库，或者可以是其它一些形式。The topical compositions of the present invention may also be delivered to the skin using conventional skin patches or devices, wherein the active ingredient composition is contained within a layered structure which is secured to the skin as a drug delivery device. In such a structure, the active ingredient combination is contained in a layer, or "reservoir", above which is an upper backing layer. The layered structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the depot contains a pharmaceutically acceptable contact adhesive substance of a polymer matrix to secure the system to the skin during delivery of the active ingredient. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. The choice of a particular polymeric binder depends on the particular active ingredient, vehicle, etc., ie, the binder must be compatible with all components of the composition containing the active ingredient. Alternatively, the reservoir containing the active ingredient and the skin contact adhesive should be separate and distinct layers with the adhesive under the reservoir, in which case the reservoir may be a polymer matrix as described above, or it may be a liquid Or a hydrogel reservoir, or it could be some other form.

这些层状物中的垫层是作为该装置的表面层，作为层状结构的主要结构元件，并给予该装置以相当多的弹性。选择作为垫层的材料应当是对所述活性成分和含有所述活性成分的组合物中的任何其它组分都基本不能渗透的，从而防止任何组分通过该装置的表面层丧失。所述垫层可以是闭合的或是不闭合的，这取决于是否希望皮肤在活性成分递送过程中水化。垫层优选由优选柔韧的弹性材料的薄片或膜组成。适用于垫层的聚合物的实例包括聚乙烯，聚丙烯和聚酯。The underlayment in these layers serves as the surface layer of the device, acts as the main structural element of the layered structure, and imparts considerable elasticity to the device. The material selected for the backing layer should be substantially impermeable to both the active ingredient and any other component of the composition containing the active ingredient, thereby preventing loss of any component through the surface layer of the device. The cushion may be occlusive or non-occlusive, depending on whether hydration of the skin during active ingredient delivery is desired. The underlayment preferably consists of a sheet or film of a preferably flexible elastic material. Examples of suitable polymers for the underlayment include polyethylene, polypropylene and polyester.

在储存过程中和使用前，所述层状结构还包括释放衬垫。立即使用前从所述装置上除去该层以使其基底面，或者是活性成分储库或者是独立的接触粘附层暴露，使得所述系统能够固定于皮肤上。所述释放衬垫应当用活性成分／载体不可透过的材料制成。During storage and prior to use, the layered structure also includes a release liner. This layer is removed from the device immediately prior to use to expose the base surface, either the active ingredient reservoir or the separate contact-adhesive layer, allowing the system to be secured to the skin. The release liner should be made of active ingredient/carrier impermeable material.

该装置可以用本领域公知的传统技术制造，例如将胶粘剂，活性成分和载体的流体混合物浇模于垫层上，然后使释放衬垫成层。类似地，可以将胶粘剂混合物浇模于释放衬垫上，然后使垫层成层。或者，可以在没有活性成分或赋形剂的情况下制备活性成分储库，然后通过“浸吸(soaking)”在活性成分／载体混合物中来载入。The device can be manufactured using conventional techniques well known in the art, such as casting the fluid mixture of adhesive, active ingredient and carrier onto the backing and then layering the release liner. Similarly, the adhesive mixture can be cast onto a release liner and the liner layered. Alternatively, active ingredient depots can be prepared without active ingredient or excipients and then loaded by "soaking" in the active ingredient/carrier mixture.

与本发明中的局部制剂一样，包含于这些层状系统的活性成分储库中的活性成分组合物可以含有多种组分。在一些情况下，所述活性成分可以以“纯(neat)”的方式递送，即不含其它的流体。但是在大部分情况下，所述活性成分溶解，分散或悬浮于合适的药学可接受的载体，典型地是溶剂或凝胶中。其它可能含有的组分包括防腐剂，稳定剂和表面活性剂等。As with the topical formulations of the present invention, the active ingredient compositions contained in the active ingredient reservoirs of these layered systems may contain a variety of components. In some cases, the active ingredient may be delivered "neat," ie, free of other fluids. In most cases, however, the active ingredient is dissolved, dispersed or suspended in a suitable pharmaceutically acceptable carrier, typically a solvent or gel. Other possible ingredients include preservatives, stabilizers and surfactants.

本文所述的药物组合物还可以包含合适的固体或凝胶相载体或赋形剂。这种载体或赋形剂的实例包括但不限于碳酸钙，磷酸钙，各种糖，淀粉，纤维素衍生物，明胶和聚合物例如聚乙二醇。The pharmaceutical compositions described herein may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycol.

剂量取决于病痛的类型，严重程度和表现形式，以及患者对于所使用的活性成分和剂型的反应，特定缀合物的效力和所使用的给药余途径。所属领域普通技术人员能容易地确定最佳的剂量，剂量给药方法和重复率。正确的配方，给药途径和剂量可以由各个医师根据患者情况选择(参见例如，Fingl，et al.，1975，in“The PharmacologicalBasis of Therapeutics”，Ch.1p.l)。Dosage will depend on the type, severity and presentation of the affliction, as well as the patient's response to the active ingredient and dosage form employed, the potency of the particular conjugate and other routes of administration utilized. Optimum dosages, dosing regimens and repetition rates can be readily determined by one of ordinary skill in the art. The correct formulation, route of administration and dosage can be selected by the individual physician according to the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

因此，依赖于要治疗的状况的严重程度和反应性，剂量可以是单次或是重复给药，治疗持续几天到几周，或者直至治愈或皮肤创伤减小。Thus, depending on the severity and responsiveness of the condition to be treated, the dosage can be a single or repeated administration, and the treatment can be continued from several days to several weeks, or until healing or skin trauma is reduced.

在本发明的某些方面中使用体内或离体(细胞)基因治疗技术，其涉及细胞转化和基因敲入类型的转化。本文所用的基因治疗指将感兴趣的遗传物质(例如DNA或RNA)转入宿主中以治疗或防止遗传性的或获得性的疾病或状况或表型。所述感兴趣的遗传物质编码希望在体内产生的产品(例如蛋白质，多肽，肽，功能性RNA，反义RNA)。例如，所述感兴趣的遗传物质可以编码具有治疗价值的激素，受体，酶，多肽或肽。其综述一般可参见“Gene Therapy”(Advanced inPharmacology40，Academic Press，1997)原文。In certain aspects of the invention in vivo or ex vivo (cell) gene therapy techniques are used, involving transformation of cells and knock-in type transformations. Gene therapy as used herein refers to the transfer of genetic material of interest (eg, DNA or RNA) into a host to treat or prevent an inherited or acquired disease or condition or phenotype. The genetic material of interest encodes a product (eg, protein, polypeptide, peptide, functional RNA, antisense RNA) that is desired to be produced in vivo. For example, the genetic material of interest may encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Its review can generally refer to the original text of "Gene Therapy" (Advanced inPharmacology 40, Academic Press, 1997).

基因治疗的两种基本方法是(1)离体；和(ii)体内基因治疗。在离体基因治疗中从患者中取出细胞或者从另一种来源衍生出细胞，培养并在体外治疗。通常，将功能性的替换基因通过合适的基因递送载体／方法(转染，转导，同源重组等)和，如果需要时，表达系统引入细胞，然后在培养物中使修饰的细胞扩增并使其返回宿主／患者中。这些遗传上重新植入的细胞已显示在原位表达转染的遗传物质。The two basic approaches to gene therapy are (1) ex vivo; and (ii) in vivo gene therapy. In ex vivo gene therapy cells are removed from the patient or derived from another source, cultured and treated outside the body. Typically, a functional replacement gene is introduced into the cells via a suitable gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and, if desired, an expression system, and the modified cells are expanded in culture and return it to the host/patient. These genetically reimplanted cells have been shown to express transfected genetic material in situ.

在体内基因治疗中，靶细胞不从受试者中取出，而是将要转移的遗传物质原位，也就是在接受体中引入接受生物体的细胞中。在另一个实施方案中，如果宿主基因是有缺陷的，所述基因在原位被修复(Culver，1998.(Abstract)Antisense DNA&RNA basedtherapeutics，February1998，Coronado，CA)。这些遗传上改变的细胞已显示在原位表达转染的遗传物质。In in vivo gene therapy, the target cells are not removed from the subject, but the genetic material to be transferred is introduced in situ, ie in the recipient, into the cells of the receiving organism. In another embodiment, if the host gene is defective, the gene is repaired in situ (Culver, 1998. (Abstract) Antisense DNA & RNA based therapeutics, February 1998, Coronado, CA). These genetically altered cells have been shown to express transfected genetic material in situ.

所述基因表达载体能够将异源核酸递送／转移到宿主细胞中。所述表达载体可以包括能以细胞选择性的方式控制所述核酸的靶定，表达和转录的元件，如本领域公知的。应当注意到通常所述基因的5’UTR和／或3’UTR可被表达载体的5’UTR和／或3’UTR取代。因此，本文所述的表达载体可以，如果需要的话，不包括要转移的实际基因的5’UTR和／或3’UTR，只包括特定的氨基酸编码区域。The gene expression vector is capable of delivering/transferring heterologous nucleic acid into host cells. The expression vector may include elements capable of controlling the targeting, expression and transcription of the nucleic acid in a cell-selective manner, as is known in the art. It should be noted that generally the 5'UTR and/or 3'UTR of the gene may be replaced by the 5'UTR and/or 3'UTR of the expression vector. Thus, the expression vectors described herein may, if desired, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred, but only the specific amino acid coding region.

所述表达载体可以包括用于控制所述异源物质的转录的启动子，所述启动子可以是组成型的或是诱导型的以允许选择性转录。任选地可以包括获得必要的转录水平所需的增强子。增强子通常是任何不翻译的DNA序列，临近编码序列起作用(以顺式)以改变启动子所支配的基本转录水平。所述表达载体还可以含有如下文所述的选择基因。The expression vector may include a promoter for controlling the transcription of the heterologous substance, which may be constitutive or inducible to allow selective transcription. Enhancers required to obtain the necessary levels of transcription can optionally be included. An enhancer is generally any untranslated DNA sequence that acts (in cis) adjacent to a coding sequence to alter the basal level of transcription directed by the promoter. The expression vector may also contain a selection gene as described below.

载体可以以本领域公知的多种方法中的任何一种引入细胞或组织。这些方法在Sambrook et al.，Molecular Cloning：A LaboratoryManual，Cold SpringsHarbor Laboratory，New York1989，1992)，Ausubel et al.，Current Protocols in Molecular Biology，JohnWiley and Sons，Baltimore，Maryland1989)，Chang et al.SomaticGene Therapy，CRC Press，Ann Arbor，MI1995)，Vega et al.，GeneTargeting，CRC Press，Ann Arbor MI(995)，Vectors：A Surveyof Molecular Cloning Vectors and Their Uses，Butterworths，Boston MA1988)和Gilboa et al.(Biotechniques4(6)：504-512，1986)中有描述，包括，例如稳定或暂时性的转染，脂质体转染(lipofection)，电穿孔和用重组病毒载体感染。此外，参见美国专利4,866,042中的涉及中枢神经系统的载体以及美国专利5,464,764和5,487,992中的正负选择方法。Vectors can be introduced into cells or tissues by any of a variety of methods known in the art. These methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York 1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland 1989), Chang et al. Somatic Gene Therapy , CRC Press, Ann Arbor, MI1995), Vega et al., GeneTargeting, CRC Press, Ann Arbor MI(995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA1988) and Gilboa et al. (Biotechniques (6): 504-512, 1986), including, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Also, see US Patent 4,866,042 for vectors involving the central nervous system and US Patents 5,464,764 and 5,487,992 for positive and negative selection methods.

通过感染引入核酸与其它所列出的方法相比具有几个优点。由于感染特性因而能够获得更高的效率。而且病毒是非常特化的，典型地可以感染并在特定的细胞类型中增殖。因而，它们天然的特异性可以用于使载体在体内或在组织中或细胞的混合培养物中靶定特异的细胞类型。病毒载体还可以用特异的受体或配体修饰以通过受体介导的事件改变靶的特异性。Introduction of nucleic acid by infection has several advantages over the other listed methods. Higher efficiencies can be achieved due to the infectious nature. And viruses are very specialized, typically infecting and multiplying in specific cell types. Thus, their natural specificity can be used to target vectors to specific cell types in vivo or in tissues or mixed cultures of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor-mediated events.

用于引入和表达重组序列的DNA病毒载体的特定实例是腺病毒衍生的载体Adenop53TK。该载体表达疱疹病毒胸苷激酶(TK)基因，以提供正向或负向选择，和所期望的重组序列的表达盒。该载体可用于感染具有腺病毒受体的细胞，包括大部分上皮来源和其它来源的组织。该载体和其它具有类似的所期望的功能的载体可用于处理混合群体的细胞，包括，例如细胞的体外或离体培养物，组织或人受试者。A specific example of a DNA viral vector for the introduction and expression of recombinant sequences is the adenovirus-derived vector Adenop53TK. The vector expresses the herpesvirus thymidine kinase (TK) gene to provide positive or negative selection, and an expression cassette for the desired recombinant sequence. This vector can be used to infect cells with adenovirus receptors, including most tissues of epithelial and other origins. This vector, and others with similar desired functions, can be used to treat mixed populations of cells, including, for example, in vitro or ex vivo cultures of cells, tissues, or human subjects.

还可以包含限制在特定细胞类型中表达的特征。这种特征包括，例如，对所希望的细胞类型特异的启动子和调控元件。Features that restrict expression to specific cell types may also be included. Such features include, for example, promoters and regulatory elements specific for the desired cell type.

此外，重组病毒载体可用于所希望的核酸的体内表达，这是由于它们可提供例如横向感染和靶定特异性的优点。横向感染是例如逆转录病毒的生命周期中固有的，是单个感染细胞产生许多子代病毒颗粒，释放出并感染相邻的细胞的过程。结果是大片区域迅速被感染，其中大部分最初并没有被原始的病毒颗粒感染。这与垂直类型的感染相反，其中感染试剂只通过下一代子代传播。也可以制造不能横向传播的病毒载体。如果目的是将特定基因引入有限数目的靶细胞的话，该特征可能是有用的。In addition, recombinant viral vectors are useful for the in vivo expression of desired nucleic acids, since they may offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, eg, retroviruses, the process by which a single infected cell produces many progeny virus particles, which are released and infect neighboring cells. The result is that large areas are rapidly infected, most of which were not initially infected by the original virus particles. This is in contrast to a vertical type of infection, where the infectious agent is only transmitted through the next generation of progeny. It is also possible to make viral vectors that cannot spread laterally. This feature may be useful if the aim is to introduce a particular gene into a limited number of target cells.

如上所述，病毒是非常特化的感染试剂，在许多情况下可以避开宿主的防御机制。典型地，病毒感染并在特定类型的细胞中增殖。病毒载体的靶定特异性是利用了它的天然的特异性，特异性靶定预定细胞类型，因而将重组基因引入感染细胞中。本发明的方法和组合物中使用的载体取决于所希望靶定的细胞类型，本领域技术人员均知道。As mentioned above, viruses are very specialized infectious agents that can in many cases evade host defense mechanisms. Typically, viruses infect and multiply in specific types of cells. The targeting specificity of a viral vector utilizes its natural specificity to specifically target a predetermined cell type, thereby introducing recombinant genes into infected cells. The vectors used in the methods and compositions of the invention depend on the type of cell desired to be targeted, as will be known to those skilled in the art.

可以构建逆转录病毒载体作为感染颗粒或只是进行起始一轮的感染。在第一种情况中，对病毒基因组进行修饰使其保持所有必需基因，调控序列和包装信号以合成新的病毒蛋白和RNA。这些分子一旦合成，宿主细胞会将所述RNA包装成新的病毒颗粒，其能进行下一轮的感染。还可以通过基因工程使所述载体的基因组编码和表达所希望的重组基因。在无感染性的病毒载体中，载体基因组通常进行突变以破坏将RNA包装成病毒颗粒所必需的病毒包装信号。没有这样的信号，任何形成的颗粒都不含有基因组，因此不能进行下一轮感染。载体的特定类型取决于预期的应用。实际的载体也是已知的，在本领域中可容易获得，或者可由本领域技术人员通过熟知的方法构建。Retroviral vectors can be constructed as infectious particles or just for the initial round of infection. In the first case, the viral genome is modified to maintain all essential genes, regulatory sequences and packaging signals for the synthesis of new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new virus particles, which are capable of the next round of infection. The genome of the vector can also encode and express the desired recombinant gene through genetic engineering. In non-infectious viral vectors, the vector genome is often mutated to disrupt viral packaging signals necessary for packaging RNA into virus particles. Without such a signal, any particles formed do not contain a genome and thus cannot proceed to the next round of infection. The particular type of carrier depends on the intended application. The actual vectors are also known, readily available in the art, or can be constructed by methods well known to those skilled in the art.

重组载体可以几种方式给药。如果使用病毒载体，例如，给药过程可以利用它们的靶特异性，因而不用在疾病部位局部给药。但是，局部给药可以更快更有效地治疗。Recombinant vectors can be administered in several ways. If viral vectors are used, for example, the administration procedure can take advantage of their target specificity, thus eliminating the need for local administration at the site of disease. However, topical administration allows for faster and more effective treatment.

包括敲入程序所用的同源重组在内的体内和离体细胞转化过程如下述文献中所述，例如，In vivo and ex vivo cell transformation procedures, including homologous recombination for knock-in procedures, are described in, for example,

                                                           UnitedStates Patents5,487,992，5,464,764，5,387,742，5,360,735，5,347,075，5,298,422，5,288,846，5,221,778，5,175,385，5,175,384，5,175,383，4,736,866以及Burkeand Olson,Methods in Enzymology,194：251-2701991)；Capecchi，Science244：1288-12921989)；Davies et al.，Nucleic Acids Research,20(11)2693-2698 1992)；Dickinson et al.，Human Molecular Genetics，2(8)：1299-13021993)；Duff andLincoln,“Insertion of a pathogenic mutation into a yeast artificial chromosomecontaining the human APP gene and expression in ES cells”，Research Advances inAlzheimer’s Disease and Related Disorders，1995；Huxley et al.,Genomics,9：742-7501991)；Jakobovits et al.,Nature,362：255-2611993)；Lamb et al.,Nature Genetics,5：22-291993)；Pearson and Choi,Proc.Natl. Acad.Sci.USA1993).90：10578-82；Rothstein,Methods in Enzymology,194：281-3011991)；Schedl et al.，Nature,362：258-2611993)；Strauss et al.，Science，259：1904-19071993).UnitedStates Patents5,487,992，5,464,764，5,387,742，5,360,735，5,347,075，5,298,422，5,288,846，5,221,778，5,175,385，5,175,384，5,175,383，4,736,866以及Burkeand Olson,Methods in Enzymology,194：251-2701991)；Capecchi，Science244：1288-12921989)； Davies et al., Nucleic Acids Research, 20(11)2693-2698 1992); Dickinson et al., Human Molecular Genetics, 2(8):1299-13021993); Duff and Lincoln, "Insertion of a pathogenic mutation into a yeast artificial chromosome containing the human APP gene and expression in ES cells”, Research Advances in Alzheimer's Disease and Related Disorders, 1995; Huxley et al., Genomics, 9: 742-750 (1991); Jakobovits et al., Nature, 362: 1595-26) ; Lamb et al., Nature Genetics, 5: 22-291993); Pearson and Choi, Proc. Natl. Acad. Sci. USA1993). 90: 10578-82; Rothstein, Methods in Enzymology, 194: 281-3011991); Schedl et al., Nature, 362:258-2611993); Strauss et al., Science, 259:1904-19071993).

进一步，专利申请WO94／23049，WO93／14200，WO94／06908，WO94／28123也提供了部分信息。Further, patent applications WO94/23049, WO93/14200, WO94/06908, WO94/28123 also provide partial information.

本领域普通技术人员可参考下述非限制性的实施例显而易见地知晓本发明的其它目的，优点和新的特征。另外，上述的和下述权利要求部分的权利要求中所述的本发明的每一个不同的实施方式和方面在下述实施例中都有实验支持。Other objects, advantages and novel features of the present invention will be apparent to those skilled in the art with reference to the following non-limiting examples. In addition, each of the various embodiments and aspects of the invention described above and in the claims in the following claims section has experimental support in the following examples.

实施例Example

参考下述实施例，与上述说明一起以非限制性的方式对本发明进行举例说明。The invention, together with the foregoing description, is illustrated in a non-limiting manner by reference to the following examples.

通常，本文所用的术语和本发明中所用的实验程序包括分子，生物化学，微生物和重组DNA技术。这些技术在文献中已有详细解释。参见，例如，Generally, the nomenclature used herein and the laboratory procedures employed in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the literature. See, for example,

                         ″Molecular Cloning：A Laboratory Manual″Sambrook etal.，(1989)；″Current Protocols in Molecular Biolo gy″Volumes I-III Ausubel，R.M.，ed.(1994)；Ausubel et al.，″Current Protocols in Molecular Biology″，JohnWiley andSons,Baltimore,Maryland(1989)；Perbal，″A Practical Guide to Molecular Cloning″，John Wiley&Sons,New York(1988)；Watson et al.，″Recombinant DNA″，ScientificAmerican Books，New York；Birren et al.(eds)″Genome Analysis：A LaboratoryManual Series″，Vols.1-4，Cold Spring Harbor Laboratory Prres，New York(1998)；"Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology" , John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. .(eds) "Genome Analysis: A Laboratory Manual Series", Vols.1-4, Cold Spring Harbor Laboratory Preres, New York (1998);

方法如method such as

                           U.S.Pat.Nos.4,666,828；4,683,202；4,801,531；5,192,659and5,272,057；″Cell Biology：A Laboratory Handbook″，Volumes I-IIICellis,J.E.，ed.(1994)；″Culture of Animal Cells-A Manual of Basic Technique″byFreshney，Wiley-Liss，N.Y.(1994)，Third Edition；″Current Protocols inImmunology″Volumes I-III Coligan J.E.，ed.(1994)；Stites et al.(eds)，″Basic andClinical Immunology″(8th Edition)，Appleton & Lange，Norwalk,CT(1994)；Mishelland Shiigi(eds)，″Selected Methods in Cellular Immunology″，W.H.Freeman andCo.，New York(1980)；U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells-A Manual of Techni que" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980);

中所述；可获得的免疫检测方法广泛描述于专利和科技文献中，参见，例如described in ; available immunoassays are widely described in the patent and scientific literature, see, e.g.

                                       U.S.Pat.Nos.3,791,932；3,839,153；3,850,752；3,850,578；3,853,987；3,867,517；3,879,262；3,901,654；3,935,074；3,984,533；3,996,345；4,034,074；4,098,876；4,879,219；5,011,771and5,281,521；″Oligonucleotide Synthesis″Gait,M.J.，ed.(1984)；“Nucleic Acid Hybridization″Hames,B.D.，and Higgins S.J.，eds.(1985)；″Transcription and Translation″Hames，B.D.，and Higgins S.J.，eds.(1984)；″Animal Cell Culture″Freshney，R.I.，ed.(1986)；″Immobilized Cells and Enzymes″IRL Press，(1986)；″A Practical Guideto Molecular Cloning″Perbal，B.，(1984)and″Methods in Enzymology″Vol.1-317，Academic Press；″PCR Protocols：A Guide To Methods And Applications″，AcademicPress，San Diego，CA(1990)；Marshak et al.，″Strategies for Protein Purification andCharacterizatio-A Laboratory Course Manual″CSHL Press(1996)；U.S.Pat.Nos.3,791,932；3,839,153；3,850,752；3,850,578；3,853,987；3,867,517；3,879,262；3,901,654；3,935,074；3,984,533；3,996,345；4,034,074；4,098,876；4,879,219；5,011,771and5,281,521；″Oligonucleotide Synthesis″Gait,M.J.，ed.(1984 ); "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., eds. (1985); "Transcription and Translation" Hames, B.D., and Higgins S.J., eds. (1984); "Animal Cell Culture" Freshney, R.I., eds. .(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterizatio-A Laboratory Course Manual", CSHL Press (1996);

在此引入所有这些文献的全文作为参考。本文中还提供了其它一般参考。其中所述程序是本领域熟知的，是为读者方便而提供的。其中包含的所有信息在此都引入作为参考。All of these documents are hereby incorporated by reference in their entirety. Other general references are also provided in this document. The programs described therein are well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

材料和实验方法Materials and Experimental Methods

材料：组织培养介质和血清购于Biological Industries(BeitHaEmek，Israel)。强化化学发光(Enhanced Chemical Luminescence，ECL)的操作使用从BioRad(Israel)购买的试剂盒。单克隆抗p-tyr的抗体购于Upstate Biotechnology Inc.(Lake Placid，NY，USA)。PKC同种型的单克隆和多克隆抗体购于Santa Cruz(California，USA)和Transduction Laboratories(Lexington，KY)。α6大鼠抗小鼠mAb(GoH3)购于Pharmingen(San Diego，CA)。α6A细胞质结构域的抗体6844得自Dr.V.Quaranta(Scr ipps Research Insti tute，La Jolla，CA)的馈赠。针对小鼠β4的细胞外结构域(346-11A)的大鼠mAb得自Dr.S.J.Kennel(Oak Ridge National Laboratory，Oak Ridge，TN)的馈赠。针对磷酸化酪氨酸的大鼠mAB购自Sigma(St.Louis，MO)，兔抗磷酸化丝氨酸购自Zymed(San Francisco，CA)。辣根过氧化物酶抗兔和抗小鼠IgG得自Bio-Rad(Israel)。亮肽素，抑肽酶，PMSF，DTT，Na-原钒酸盐，和抑胃肽购自Sigma Chemicals(St.Louis，MO)。胰岛素(humulin R-重组人胰岛素)购自EliLilly FranceSA(Fergersheim，France)。IGF1得自Cytolab(Israel)的馈赠。角蛋白14抗体购自Babco-Convance(Richmond，CA)。BDGF-BB购自R&D系统(Minneapolis)，十四酰化(myristolated)的PKCα伪底物购自Calbinochem(San Diego，CA)。Materials: Tissue culture media and serum were purchased from Biological Industries (Beit HaEmek, Israel). The operation of Enhanced Chemical Luminescence (ECL) used a kit purchased from BioRad (Israel). Monoclonal anti-p-tyr antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Monoclonal and polyclonal antibodies to PKC isotypes were purchased from Santa Cruz (California, USA) and Transduction Laboratories (Lexington, KY). α6 rat anti-mouse mAb (GoH3) was purchased from Pharmingen (San Diego, CA). Antibody 6844 to the α6A cytoplasmic domain was obtained from a gift of Dr. V. Quaranta (Scripps Research Institute, La Jolla, CA). A rat mAb directed against the extracellular domain of mouse β4 (346-11A) was obtained from a gift of Dr. S.J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN). Rat mAb against phosphotyrosine was purchased from Sigma (St. Louis, MO) and rabbit anti-phosphoserine was purchased from Zymed (San Francisco, CA). Horseradish peroxidase anti-rabbit and anti-mouse IgG were obtained from Bio-Rad (Israel). Leupeptin, aprotinin, PMSF, DTT, Na-orthovanadate, and gastric inhibitory peptide were purchased from Sigma Chemicals (St. Louis, MO). Insulin (humulin R-recombinant human insulin) was purchased from Eli Lilly France SA (Fergersheim, France). IGF1 was obtained from a gift from Cytolab (Israel). Keratin 14 antibody was purchased from Babco-Convance (Richmond, CA). BDGF-BB was purchased from R&D Systems (Minneapolis) and myristolated PKCα pseudosubstrate was purchased from Calbinochem (San Diego, CA).

鼠角质化细胞的分离和培养：如文献中所述从新生皮肤上分离原代角质化细胞(18)。在含有8％的Chelex(Chelex-100，BioRad)处理的胎牛血清的Eagle’s Minimal Essential Medium(EMEM)中培养角质化细胞。为了维持增殖的基底细胞的表型，将最终的Ca²⁺浓度调节为0.05mM。铺板后五天到七天进行实验。Isolation and culture of murine keratinocytes: Primary keratinocytes were isolated from neonatal skin as described in the literature (18). Keratinocytes were cultured in Eagle's Minimal Essential Medium (EMEM) containing 8% Chelex (Chelex-100, BioRad)-treated fetal bovine serum. To maintain the phenotype of proliferating basal cells, the final Ca2⁺ concentration was adjusted to 0.05 mM. Experiments were performed five to seven days after plating.

细胞提取物的制备和Western blot分析：为得到膜组分粗提物，在含有10μg／ml抑肽酶，10μg／ml亮肽素，2μg／ml抑胃肽(pepstat in)，1mMPMSF，10mM EDTA，200μMNaVO₄和10mM NaF的PBS中破碎细胞，制备全细胞溶解物。经过匀浆和4次冻／溶循环后，将溶解物置于微型离心机中以最大速度在4℃旋转20分钟。将含有可溶性细胞溶质蛋白组分的上清液转移至另一个管子中。将沉淀重悬于250μl含有1％Tr iton X-100以及蛋白酶和磷酸酶抑制剂的PBS中，在4℃保温30分钟，在4℃在微型离心机中以最大速度旋转。上清中含有膜组分。用改进的Lowery检测(Bio-Rad DC Protein Assay Kit)测定蛋白质浓度。如文献中所述对细胞蛋白质组分进行Western blot分析(6)。Preparation of cell extracts and Western blot analysis: In order to obtain crude extracts of membrane fractions, in the presence of 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin, 1 mMPMSF, 10 mM EDTA , 200 μM NaVO₄ and 10 mM NaF in PBS to disrupt cells to prepare whole cell lysates. After homogenization and 4 freeze/thaw cycles, the lysate was spun in a microcentrifuge at maximum speed for 20 minutes at 4°C. Transfer the supernatant containing the soluble cytosolic protein fraction to another tube. The pellet was resuspended in 250 μl of PBS containing 1% Triton X-100 and protease and phosphatase inhibitors, incubated at 4°C for 30 minutes, and spun at maximum speed in a microcentrifuge at 4°C. The supernatant contains membrane fractions. Protein concentrations were determined with a modified Lowery assay (Bio-Rad DC Protein Assay Kit). Western blot analysis of cellular protein fractions was performed as described in the literature (6).

用于免疫沉淀的细胞溶解物的制备：含有角质化细胞的培养皿用不含Ca²⁺／Mg²⁺的PBS洗涤。用机械力使细胞分离，置于含有蛋白酶和磷酸酶抑制剂的混合物(20μg／ml亮肽素，10μg／ml抑肽酶；0.1mMPMSF；1mM DTT；200μM原钒酸盐；2μg／ml抑胃肽)的RIPA缓冲液(50mM Tris·HCl pH7.4；150mM NaCl；1mM EDTA；10mM NaF；1％Triton x100；0.1％SDS，1％脱氧胆酸钠)中。将此制备物在4℃在微型离心机中以最大速度离心20分钟。将上清液用于免疫沉淀。Preparation of cell lysates for immunoprecipitation: Petri dishes containing keratinocytes were washed with PBS without Ca²⁺ /Mg²⁺ . The cells were detached by mechanical force and placed in a mixture containing protease and phosphatase inhibitors (20 μg/ml leupeptin, 10 μg/ml aprotinin; 0.1 mMPMSF; 1 mM DTT; 200 μM orthovanadate; 2 μg/ml inhibin peptide) in RIPA buffer (50 mM Tris·HCl pH7.4; 150 mM NaCl; 1 mM EDTA; 10 mM NaF; 1% Triton x100; 0.1% SDS, 1% sodium deoxycholate). The preparation was centrifuged at maximum speed in a microcentrifuge for 20 minutes at 4°C. The supernatant was used for immunoprecipitation.

免疫沉淀：将300μg细胞溶解物与25μl蛋白A／G Sepharose(Santa Cruz，CA，USA)混合，将悬液在4℃连续旋转30分钟，进行溶解物的预处理。然后将制备物在4℃以最大速度离心10分钟，向上清液中加入30μl A／G Sepharose以及针对个别抗原的特异性多克隆或单克隆抗体(稀释率1：100)。将样品在4℃旋转过夜。然后将悬液在4℃以最大速度离心10分钟，将沉淀用RIPA缓冲液洗涤。然后将悬液以15,000xg再次离心(4℃，10分钟)，用TBST洗涤四次。加入样品缓冲液(0.5M Tris·HCl pH6.8；10％SDS；10％甘油；4％2-β-巯基乙醇；0.05％溴酚蓝)，将样品煮沸5分钟，然后进行SDS-PAGE。Immunoprecipitation: 300 μg of cell lysates were mixed with 25 μl of protein A/G Sepharose (Santa Cruz, CA, USA), and the suspension was continuously rotated at 4°C for 30 minutes to perform pretreatment of the lysates. The preparation was then centrifuged at maximum speed for 10 minutes at 4°C, and 30 μl of A/G Sepharose and specific polyclonal or monoclonal antibodies against individual antigens (dilution ratio 1:100) were added to the supernatant. Samples were rotated overnight at 4°C. The suspension was then centrifuged at maximum speed for 10 minutes at 4°C, and the pellet was washed with RIPA buffer. The suspension was then centrifuged again at 15,000 xg (4°C, 10 minutes) and washed four times with TBST. Add sample buffer (0.5M Tris·HCl pH6.8; 10% SDS; 10% glycerol; 4% 2-β-mercaptoethanol; 0.05% bromophenol blue), boil the sample for 5 minutes, and then perform SDS-PAGE.

附着检测：用PBS中的20μg／ml的基质蛋白对二十四孔皮氏培养皿(petri plates)(Greiner)进行涂层(250μl／孔)，37℃保温1小时。保温后洗涤培养皿，并在室温与0.1％BSA培育30分钟以阻断非特异性结合。角质化细胞培养物用0.25％的胰蛋白酶短暂消化，待分离后将细胞重悬，将角质化细胞(1x10⁶)加到已涂层的孔中，在37℃培育1小时。除去未附着的细胞，将孔用PBS漂洗两次，将剩余的细胞用1M NaOH提取。用改进的Lowery检测(Bio-Rad DC ProteinAssay Kit)通过蛋白浓度确定细胞计数。结果计算为相对于未处理对照的百分数。Attachment Assay: Twenty-four well petri plates (Greiner) were coated with 20 μg/ml matrix protein in PBS (250 μl/well) and incubated at 37° C. for 1 hour. Dishes were washed after incubation and incubated with 0.1% BSA for 30 minutes at room temperature to block non-specific binding. The keratinocyte culture was briefly digested with 0.25% trypsin, and after separation, the cells were resuspended, and keratinocytes (1×10⁶ ) were added to the coated wells, and incubated at 37° C. for 1 hour. Unattached cells were removed, the wells were rinsed twice with PBS, and the remaining cells were extracted with 1M NaOH. Cell counts were determined by protein concentration using a modified Lowery assay (Bio-Rad DC Protein Assay Kit). Results are calculated as percentages relative to untreated controls.

免疫荧光：将原代角质化细胞接种于层粘连蛋白(laminin)5涂布的玻璃载玻片上。用PKC腺病毒感染两天大的角质化细胞一小时，用PBS洗涤两次，保存在低Ca²⁺浓度的MEM培养物中。感染后二十四小时；将细胞用4％多聚甲醛固定30分钟，然后用0.2％Triton透化(permeabilization)5分钟。为进行分析，将对照和PKC感染的角质化细胞用PBS漂洗，与稀释在含有1％BSA的PBS中的PKC抗体(SantaCruz)在4℃培育过夜。培育后，载玻片用PBS洗涤两次10分钟，与生物素化的抗兔的二抗培育20分钟，用PBS洗涤两次，与strepayidin-FITC培育20分钟。为分析α6β4的染色，将玻璃载玻片用0.2％Triton X-100在冰上处理5分钟，然后在甲醇中固定5分钟。将载玻片与抗α6或抗β4的抗体培育过夜，然后分别与生物素化的抗兔的二抗培育20分钟，用PBS洗涤两次，与strepavidin-FITC培育20分钟。用PBS洗涤两次后，用含有1％对苯二胺(Digma)的甘油缓冲液处理(mount)载玻片，用激光扫描共聚焦成像显微镜(MRC1024，Bio-Rad，UK)检测荧光。Immunofluorescence: Primary keratinocytes were seeded on laminin 5-coated glass slides. Two-day-old keratinocytes were infected with PKC adenovirus for one hour, washed twice with PBS, and kept in MEM cultures at low Ca^{concentrations} . Twenty-four hours post-infection; cells were fixed with 4% paraformaldehyde for 30 minutes and then permeabilized with 0.2% Triton for 5 minutes. For analysis, control and PKC-infected keratinocytes were rinsed with PBS and incubated overnight at 4°C with PKC antibody (Santa Cruz) diluted in PBS containing 1% BSA. After incubation, slides were washed twice with PBS for 10 minutes, incubated with biotinylated anti-rabbit secondary antibody for 20 minutes, washed twice with PBS, and incubated with strepayidin-FITC for 20 minutes. For analysis of α6β4 staining, glass slides were treated with 0.2% Triton X-100 for 5 minutes on ice and then fixed in methanol for 5 minutes. Slides were incubated overnight with anti-α6 or anti-β4 antibodies, then incubated with biotinylated anti-rabbit secondary antibody for 20 minutes, washed twice with PBS, and incubated with strepavidin-FITC for 20 minutes. After washing twice with PBS, slides were treated (mounted) with glycerol buffer containing 1% p-phenylenediamine (Digma), and fluorescence was detected with a laser scanning confocal imaging microscope (MRC1024, Bio-Rad, UK).

腺病毒构建体：重组腺病毒载体如文献所述构建(19)。小鼠PKC的显性失活突变体是通过用丙氨酸取代ATP结合位点的赖氨酸残基产生的。用EcoRI从SRD表达载体上切下突变体的cDNA，连接到pAxCA1w粘粒盒上，构建Ax载体。其自体磷酸化活性的破坏证明了所述基因的显性失活活性。Adenoviral constructs: Recombinant adenoviral vectors were constructed as described (19). A dominant negative mutant of mouse PKC was generated by substituting alanine for a lysine residue in the ATP-binding site. The mutant cDNA was excised from the SRD expression vector with EcoRI and ligated to the pAxCA1w cosmid cassette to construct the Ax vector. Disruption of its autophosphorylation activity demonstrates the dominant negative activity of the gene.

用PKC同种型基因转导角质化细胞：吸出培养基，用含有PKC重组腺病毒的病毒上清液感染角质化细胞培养物一小时。然后用MEM洗涤所述培养物两次并重新培养。感染后十小时，将细胞转移至不含血清的低Ca²⁺的MEM中24小时。来自对照的和用胰岛素处理的或用IGF1处理的培养物的角质化细胞用于进行增殖检测，⁸⁶Rb吸收，或者提取并分级成细胞溶质和膜组分进行免疫沉淀，免疫荧光和Westernblotting。Transduction of keratinocytes with PKC isoform genes: Aspirate the medium and infect keratinocyte cultures with viral supernatant containing PKC recombinant adenovirus for one hour. The cultures were then washed twice with MEM and regrown. Ten hours after infection, cells were transferred to serum-free low Ca2⁺ MEM for 24 hours. Keratinocytes from control and insulin-treated or IGF1-treated cultures were used for proliferation assays,⁸⁶ Rb uptake, or extracted and fractionated into cytosolic and membrane fractions for immunoprecipitation, immunofluorescence and Western blotting.

PKC活性：在用经适当处理后的角质化细胞培养物新鲜制备的免疫沉淀物中测定特异的PKC活性。这些细胞溶解物制备于不含NaF的RIPA缓冲液中。用SigmaTECT Protein Kinase C Assay System(Promega，Madison，WI，USA)测定活性，根据制造商的说明操作。在这些研究中用PKCα伪底物作为底物。PKC activity: Specific PKC activity was determined in freshly prepared immunoprecipitates from appropriately treated keratinocyte cultures. These cell lysates were prepared in NaF-free RIPA buffer. Activity was determined with the SigmaTECT Protein Kinase C Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. A PKCα pseudosubstrate was used as the substrate in these studies.

细胞增殖：在24孔板中用[³H]胸苷掺入测定细胞增殖。细胞用[³H]胸苷(1μCi／ml)脉冲过夜。培育后细胞用PBS洗涤五次，将5％TCA加入每个孔30分钟。除去溶液，将细胞溶于1％Triton X-100中。在Tricarb液体闪烁计数器的³H窗口中对掺入细胞的标记的胸苷进行计数。Cell proliferation: Cell proliferation was assayed by [³ H]thymidine incorporation in 24-well plates. Cells were pulsed overnight with [³ H]thymidine (1 μCi/ml). After incubation the cells were washed five times with PBS and 5% TCA was added to each well for 30 minutes. The solution was removed and the cells were dissolved in 1% Triton X-100. Incorporation of labeled thymidine into cells was counted in the³ H window of a Tricarb liquid scintillation counter.

Na⁺／K⁺泵活性：Na⁺／K⁺泵活性通过测量全细胞在1ml含有2mM RbCl和2.5μCi的⁸⁶Rb的并且不含K⁺的PBS中对⁸⁶Rb的乌巴因敏感(ouabain-sensitive)的吸收(uptake)来确定。15分钟后吸去培养液终止Rb的吸收，然后将细胞在冷的4℃的不含K⁺的PBS中快速漂洗四次，并溶于1％Triton X-100中。在闪烁瓶中将培养皿上的细胞加入到3ml H₂O中。在Tricarb液体闪烁计数器的³H窗口中对样品进行计数。特定地与Na⁺／K⁺泵活性相关的Rb吸收是通过将在缺乏抑制剂的条件下测定的吸收值减去在存在10^-4M乌巴因的条件下积累的cpm来确定。^Na+ /K⁺ pump^activity : Na⁺ /K⁺ pump activity was measured by measuring the sensitivity of whole cells to⁸⁶ Rb oubain (ouabain- Sensitive) absorption (uptake) to determine. After 15 minutes, the medium was aspirated to terminate Rb uptake, and the cells were quickly rinsed four times in cold 4°C K⁺ -free PBS and dissolved in 1% Triton X-100. Add the cells on the dish to 3 ml_H2O in a scintillation vial. Samples were counted in the³ H window of a Tricarb liquid scintillation counter. Rb uptake specifically associated with Na⁺ /K⁺ pump activity was determined by subtracting the cpm accumulated in the presence of 10^-4 M ouabain from the uptake measured in the absence of inhibitor.

PKC免疫激酶检测：纯化的和标准化的PKC同工酶由Dr.P.Blumberg(NCI，NIH，U.S.)和Dr.Marcello G.Kazanietz(University of Pennsylvania，School of Medicine)慷慨提供。收集原代角质化细胞于500μl1％Triton Lysis缓冲液(1％TritonX-100，10μg／ml抑肽酶和亮肽素，2μg／ml抑胃肽，1mM PMSF，1mMEDTA，200μM Na₂VO₄，10mM NaF在1x PBS中)中。胞溶产物在4℃培育30分钟，在4℃以16,000xg旋转30分钟。将上清液转至新鲜管中。用5μg／个样品的抗α6／GoH3(PharMingen)和30μl／个样品的蛋白A／G-plus agarose slurry(Santa Cruz)在4℃对细胞溶解物进行免疫沉淀过夜。珠子用RIPA缓冲液洗涤一次，用50mM Tris／HClpH7.5洗涤两次。向每个检测中加入35μl反应缓冲液(1mM CaCl₂，20mM MgCl₂，50mM Tris·HCl pH7.5)。对于每个检测，向淤浆中加入5.5μl／个检测的含有DMSO或10mM TPA的磷脂囊泡悬液，以及标准化量的特异PKC同工酶。加入10μl／个检测的125mM ATP(1.25μCi／个检测[γ-32P]ATP，Amersham)起始反应，在30℃继续10分钟。珠子用RIPA缓冲液洗涤两次。加入30μl／个样品的蛋白质加样染料(protein loading dye)(3x Laemmli，5％SDS)，将样品在水浴中煮沸5分钟。用SDS-PAGE在8.5％的凝胶上分离蛋白质，转移到Protran膜(Schleicher&Schuell)上，用放射自显影显像。组蛋白的磷酸化和PKC底物肽的磷酸化用作PKC活性的对照。PKC immunokinase assay: Purified and standardized PKC isozymes were generously provided by Dr. P. Blumberg (NCI, NIH, US) and Dr. Marcello G. Kazanietz (University of Pennsylvania, School of Medicine). Collect primary keratinocytes in 500μl 1% Triton Lysis buffer (1% TritonX-100, 10 μg/ml aprotinin and leupeptin, 2 μg/ml gastric inhibitory peptide, 1 mM PMSF, 1 mM EDTA, 200 μM Na₂ VO₄ , 10 mM NaF in 1x PBS). Lysates were incubated at 4°C for 30 minutes and spun at 16,000 xg for 30 minutes at 4°C. Transfer the supernatant to a fresh tube. Cell lysates were immunoprecipitated overnight at 4°C with 5 μg/sample of anti-α6/GoH3 (PharMingen) and 30 μl/sample of protein A/G-plus agarose slurry (Santa Cruz). Beads were washed once with RIPA buffer and twice with 50 mM Tris/HCl pH 7.5. 35 μl of reaction buffer (1 mM CaCl₂ , 20 mM MgCl₂ , 50 mM Tris·HCl pH 7.5) was added to each assay. For each assay, 5.5 μl/assay of a phospholipid vesicle suspension containing DMSO or 10 mM TPA was added to the slurry, along with standardized amounts of specific PKC isozymes. Reactions were initiated by adding 10 μl/assay of 125 mM ATP (1.25 μCi/assay [γ-32P]ATP, Amersham) and continued at 30° C. for 10 minutes. Beads were washed twice with RIPA buffer. 30 μl/sample of protein loading dye (3x Laemmli, 5% SDS) was added and the samples were boiled in a water bath for 5 minutes. Proteins were separated on 8.5% gels by SDS-PAGE, transferred to Protran membranes (Schleicher & Schuell) and visualized by autoradiography. Phosphorylation of histones and phosphorylation of PKC substrate peptides were used as controls for PKC activity.

实验结果Experimental results

实施例1Example 1

用重组腺病毒载体有效过表达PKC同种型Efficient overexpression of PKC isoforms with recombinant adenoviral vectors

用重组β-半乳糖苷酶腺病毒获得高的感染率，超过90％的培养的角质化细胞群体表达重组蛋白。重组β-半乳糖苷酶腺病毒感染不会影响细胞的生存力或细胞生长。进一步，β-半乳糖苷酶的表达持续长达两周的培养，在下面的实验中可用作对照感染。检测了重组PKC腺病毒构建体诱导蛋白表达和在小鼠角质化细胞培养物中正确活化的效率。如图1中的Western blotting所示，用重组PKC腺病毒构建体进行1小时的感染之后24小时，观察到特定PKC蛋白表达显著增加，是该特定同种型内源表达水平的五到十倍。早在感染后6小时在感染的角质化细胞培养物中就可检测到重组蛋白，表达的峰值在24小时获得。在整个培养阶段(长达十四天)中蛋白都持续表达。High infection rates were obtained with recombinant β-galactosidase adenovirus, with over 90% of the cultured keratinocyte population expressing the recombinant protein. Recombinant β-galactosidase adenovirus infection did not affect cell viability or cell growth. Further, expression of β-galactosidase persists for up to two weeks in culture and can be used as a control infection in the following experiments. The efficiency of recombinant PKC adenoviral constructs to induce protein expression and correct activation in mouse keratinocyte cultures was examined. As shown by Western blotting in Figure 1, 24 hours after 1 hour of infection with a recombinant PKC adenoviral construct, a significant increase in expression of a specific PKC protein was observed, five to ten-fold higher than the endogenous expression level of that specific isoform . Recombinant protein was detectable in infected keratinocyte cultures as early as 6 hours post-infection, with peak expression attained at 24 hours. Protein expression persisted throughout the culture period (up to fourteen days).

实施例2Example 2

过表达的PKC同种型被PKC活化剂激活Overexpressed PKC isoforms are activated by PKC activators

PKC同种型的重组蛋白典型地对PKC活化剂具有反应。如图2所示，用苔藓虫素1处理诱导PKCα和δ蛋白向膜组分迁移，而对PKCη和ζ同种型效果较弱，用内源同种型也得到相似结果，正如根据它们对辅因子的需要所预料的那样。Recombinant proteins of PKC isoforms are typically responsive to PKC activators. As shown in Figure 2, treatment withbryostatin 1 induced the migration of PKCα and δ proteins to the membrane fraction, whereas the effect on the PKCη and ζ isoforms was weaker, and similar results were obtained with endogenous isoforms, as indicated by their Cofactor requirements are expected.

实施例3Example 3

过表达的PKC同种型的天然形式是有活性的Native forms of overexpressed PKC isoforms are active

早在感染后18个小时，PKC激酶检测就表明不同PKC同种型的免疫沉淀物不需要进一步用PKC活化剂激活就具有酶促活性(图3)。As early as 18 hours post-infection, PKC kinase assays indicated that immunoprecipitates of different PKC isoforms were enzymatically active without further activation with PKC activators (Fig. 3).

实施例4Example 4

过表达的特定PKC同种型在原代角质化细胞中诱导不同的形态变化Overexpressed specific PKC isoforms induce distinct morphological changes in primary keratinocytes

使用的每一种PKC腺病毒构建体在原代角质化细胞中均诱导特定的形态变化(图4)。未感染的原代小鼠角质化细胞培养物和β-半乳糖苷酶感染的细胞具有培养物中的增殖的基底细胞的典型的立方形态特征。无论同种型的特异性如何，所有的PKC过表达角质化细胞都表现出了PKC活化的典型的形态变化，包括细胞伸长，出现神经元样的突起。但是，PKC同种型中的每一个对于角质化细胞形态都具有独特的效果。PKCα感染诱导角质化细胞成层，具有典型的扁平形态。相反，PKCη表现为紧缩的细胞克隆，是快速增殖的基底细胞的形态特征(图4)。所述同种型中的两个似乎影响细胞基质以及细胞和细胞间的联系。在PKCδ感染后18-48小时，细胞伸长，伸出神经元样的突起。然后是细胞逐渐从培养皿中丧失，这是在培养过程中逐渐发生的。过表达PKCζ的角质化细胞表现为圆形的角质化细胞簇，其松散地附着于培养皿上，在感染后几天逐渐地丧失。Each of the PKC adenoviral constructs used induced specific morphological changes in primary keratinocytes (Figure 4). Uninfected primary mouse keratinocyte cultures and β-galactosidase-infected cells had the typical cuboidal morphology of proliferating basal cells in culture. Regardless of isotype specificity, all PKC-overexpressing keratinocytes displayed morphological changes typical of PKC activation, including cell elongation and the appearance of neuron-like protrusions. However, each of the PKC isoforms has unique effects on keratinocyte morphology. PKCα infection induces stratification of keratinocytes with a typical flattened morphology. In contrast, PKCη manifested as compact cell clones, a morphological characteristic of rapidly proliferating basal cells (Fig. 4). Two of the isoforms appear to affect the cellular matrix and cell-to-cell connections. 18-48 hours after PKCδ infection, the cells elongate and project neuron-like processes. Then there is the gradual loss of cells from the dish, which happens gradually during the culture process. Keratinocytes overexpressing PKCζ appear as round clusters of keratinocytes that are loosely attached to the dish and are gradually lost several days after infection.

实施例5Example 5

在感染的原代角质化细胞中过表达的PKC同种型的不同定位Differential localization of overexpressed PKC isoforms in infected primary keratinocytes

由免疫荧光分析证明不同的形态变化与不同的细胞定位有关。在增殖的角质化细胞中，PKCα，PKCδ和PKCζ在细胞质中以及质膜上表达。与内源蛋白表达类似，PKCη同种型定位于角质化细胞的细胞核周区域(图5)。PKCδ和PKCζ与分布的动态变化有关，其中在细胞分离之后，PKC同种型表达主要定位于细胞膜(图5)。Different morphological changes were associated with different cellular localizations as evidenced by immunofluorescence analysis. In proliferating keratinocytes, PKCα, PKCδ and PKCζ are expressed in the cytoplasm as well as on the plasma membrane. Similar to endogenous protein expression, the PKCn isoform localized to the perinuclear region of keratinocytes (Figure 5). PKCδ and PKCζ were associated with dynamic changes in distribution, where after cell isolation, PKC isoform expression was predominantly localized to the cell membrane (Fig. 5).

实施例6Example 6

PKC同种型对α6β4表达的调控Regulation of α6β4 expression by PKC isoforms

实验结果Experimental results

检测了特定PKC同种型调节那些对于增殖的基底层的基底表型特征性的蛋白的能力。由于α6β4整联蛋白的下调是角质化细胞分化过程中发生的早期事件之一，评估了不同PKC同种型调节α6β4整联蛋白，一种特别定位于基底层的半桥粒的整联蛋白，的表达的能力。由图6的免疫杂交可看出，与对照角质化细胞中α6β4整联蛋白亚基水平相比，只有PKCδ和PKCζ同种型能够下调α6β4表达。同时，α3或β1整联蛋白亚基的水平没有减少。相反，一致地，PKCα同种型的过表达导致α6β4水平增大到对照表达的两到三倍(图6)。PKCη的过表达不影响α6β4蛋白的表达。要分化的细胞与几个特征有关，其在α6β4蛋白下调后，包括增殖速度的降低，新的角蛋白的合成，细胞分离，丧失与基底膜组分的附着。没有观察到不同PKC同种型的过表达导致角蛋白表达的变化。这包括K5和K14，基底增殖角质化细胞的特征物，和K1和K10，棘状分化(spinous differentiation)早期的特征物，的表达。此外，当用³H-胸苷掺入分析增殖速度的时候，在α6β4表达丧失和增殖潜力之间没有相关性。The ability of specific PKC isoforms to modulate those proteins that are characteristic of the basal phenotype of the proliferating basal lamina was examined. Since the downregulation of α6β4 integrin is one of the early events that occur during keratinocyte differentiation, different PKC isoforms were assessed to regulate α6β4 integrin, an integrin specifically localized to the hemidesmosomes of the basal lamina, ability to express. As can be seen from the immunoblotting in Figure 6, only the PKCδ and PKCζ isoforms were able to downregulate α6β4 expression compared to α6β4 integrin subunit levels in control keratinocytes. At the same time, the levels of α3 or β1 integrin subunits were not reduced. In contrast, consistently, overexpression of the PKCα isoform resulted in an increase in α6β4 levels to two to three-fold that of control expression ( FIG. 6 ). Overexpression of PKCη did not affect the expression of α6β4 protein. Cells to differentiate are associated with several features that, following downregulation of the α6β4 protein, include decreased proliferation rate, synthesis of new keratin, cell detachment, loss of attachment to basement membrane components. Overexpression of different PKC isoforms resulted in no changes in keratin expression were observed. This included the expression of K5 and K14, characteristics of basal proliferating keratinocytes, and K1 and K10, characteristics of early stages of spinous differentiation. Furthermore, there was no correlation between loss of α6β4 expression and proliferation potential when the rate of proliferation was analyzed by³ H-thymidine incorporation.

实施例7Example 7

过表达的PKCη和PKCδ在体外诱导角质化细胞增殖Overexpressed PKCη and PKCδ induce keratinocyte proliferation in vitro

PKCη和PKCδ的过表达分别显著诱导角质化细胞以对照五倍和两倍的水平增殖(图7)。PKCζ和PKCα不会影响细胞增殖。Overexpression of PKCη and PKCδ significantly induced keratinocyte proliferation at five-fold and two-fold levels of control, respectively (Fig. 7). PKCζ and PKCα did not affect cell proliferation.

实施例8Example 8

过表达的PKCδ和ζ在体外诱导角质化细胞分离Overexpressed PKCδ and ζ induce keratinocyte isolation in vitro

研究了PKCδ和ζ过表达的角质化细胞的附着特性。与对照角质化细胞相比，与特定的基质蛋白包括层粘连蛋白1，层粘连蛋白5，纤连蛋白和胶原蛋白的附着能力没有变化(数据未显示)。但是，在过表达PKCδ和PKCζ同种型的细胞中，细胞与培养皿之间的接触的丧失与角质化细胞逐渐从培养皿上的分离有关(图4)。The attachment properties of PKCδ and ζ overexpressed keratinocytes were investigated. The ability to attach to specific matrixproteins including laminin 1,laminin 5, fibronectin and collagen was unchanged compared to control keratinocytes (data not shown). However, in cells overexpressing the PKCδ and PKCζ isoforms, loss of cell-dish contact was associated with gradual detachment of keratinocytes from the dish (Figure 4).

实施例9Example 9

PKC同种型的过表达对α6β4整联蛋白的半桥粒定位的影响Effect of overexpression of PKC isoforms on hemidesmosomal localization of α6β4 integrins

由于α6β4表达对于半桥粒附着复合体的形成是必需的，检测了α6β4下调以及细胞分离与α6β4向半桥粒的定位之间的关系。图8显示了α6β4与半桥粒复合体之间的关系的免疫荧光分析。如图8所示，与对照感染角质化细胞相比，在过表达PKCα的角质化细胞(图6)中α6β4整联蛋白表达的上调与α6β4向半桥粒复合体整合的增加有关。过表达PKCη的细胞也诱导α6β4整联蛋白与半桥粒复合体的结合，虽然比过表达PKCα的细胞中观察到的少。正如所料，在PKCδ和PKCζ过表达的角质化细胞中α6β4整联蛋白的显著下调与α6β4向细胞半桥粒复合体整合的减少有关(图8)。这些结果表明α6β4整联蛋白在细胞-基质联系和角质化细胞向下面的基底膜的锚定中起着重要作用。而且，PKCδ和ζ介导的α6β4的下调，使角质化细胞开始以一种不同于角质化细胞分化过程的途径分离。最后，为了将PKC介导的α6β4下调，减少的半桥粒上的α6β4的整合和特定的形态变化与角质化细胞的分离联系起来，检测了在培养期间附着和分离的过表达不同PKC同种型的细胞的量的变化。在图9中，在PKC腺病毒感染后24和48小时对培养物中附着的细胞进行计数。能清楚观察到PKCδ和PKCζ在体外诱导细胞丧失。同时，培养物细胞的丧失与培养基中漂浮的细胞的增加有关。这些结果表明PKCδ和PKCζ对于控制与细胞分化和迁移早期相关的分离步骤非常重要。Since α6β4 expression is essential for hemidesmosome attachment complex formation, the relationship between α6β4 downregulation and cell detachment and localization of α6β4 to hemidesmosomes was examined. Figure 8 shows an immunofluorescence analysis of the relationship between α6β4 and the hemidesmosome complex. As shown in Figure 8, upregulation of α6β4 integrin expression in PKCα-overexpressing keratinocytes (Figure 6) was associated with increased integration of α6β4 into the hemidesmosome complex compared to control infected keratinocytes. Cells overexpressing PKCη also induced association of α6β4 integrin to the hemidesmosome complex, although less than that observed in cells overexpressing PKCα. As expected, significant downregulation of α6β4 integrin in PKCδ and PKCζ overexpressing keratinocytes was associated with reduced integration of α6β4 into cellular hemidesmosome complexes (Fig. 8). These results suggest that α6β4 integrins play an important role in cell-matrix communication and anchoring of keratinocytes to the underlying basement membrane. Moreover, PKCδ- and ζ-mediated downregulation of α6β4 initiates keratinocyte segregation in a pathway distinct from the keratinocyte differentiation process. Finally, to link PKC-mediated downregulation of α6β4, reduced integration of α6β4 on hemidesmosomes and specific morphological changes to keratinocyte detachment, overexpressing different PKC isotypes attached and detached during culture were examined. A change in the amount of cells of the type. In Figure 9, the attached cells in the culture were counted 24 and 48 hours after PKC adenovirus infection. It can be clearly observed that PKCδ and PKCζ induce cell loss in vitro. At the same time, the loss of culture cells was associated with the increase of floating cells in the medium. These results suggest that PKCδ and PKCζ are important for controlling segregation steps associated with early stages of cell differentiation and migration.

实施例10Example 10

PKCη在生理条件下有差别地调控角质化细胞的增殖和分化。PKCη differentially regulates the proliferation and differentiation of keratinocytes under physiological conditions.

从图7中可明显看出，过表达PKCη同种型的细胞以加速的速度增殖，为对照的未感染细胞五到七倍，也比过表达其它PKC同种型的角质化细胞培养物高。但是增殖的诱导取决于角质化细胞的分化状态，这是通过调节培养基中Ca²⁺的浓度决定的。在保持在低Ca²⁺浓度(0.05mM)的增殖的角质化细胞中内源PKCη定位于大多数增殖细胞的细胞核周区域(图10)。在这些条件下，PKCη的过表达诱导角质化细胞增殖的显著增加(图11)。但是，当将Ca²⁺浓度增加到0.12mM使角质化细胞分化的时候，PKCη的过表达并不诱导增殖而是进一步刺激角质化细胞分化。这些结果表明过表达PKCη仅在进行生理性增殖的细胞中诱导增殖，而并不干涉细胞的分化。在体内也观察到PKCη表达的调控作用的差异。在积极增殖的皮肤以及胚胎神经元细胞中鉴别到PKCη的表达，而在成熟的成人脑部没有观察到PKCη，在表皮中PKCη定位到皮肤的粒层。As is evident from Figure 7, cells overexpressing a PKCn isoform proliferated at an accelerated rate, five to seven times that of control, uninfected cells, and also higher than that of keratinocyte cultures overexpressing other PKC isoforms. . But the induction of proliferation depends on the differentiation state of keratinocytes, which is determined by adjusting the concentration of Ca2⁺ in the medium. In proliferating keratinocytes maintained at low Ca2⁺ concentrations (0.05 mM), endogenous PKCn localized to the perinuclear region of most proliferating cells (Figure 10). Under these conditions, overexpression of PKCn induced a marked increase in keratinocyte proliferation (Figure 11). However, when increasing the Ca²⁺ concentration to 0.12 mM to differentiate keratinocytes, overexpression of PKCη did not induce proliferation but further stimulated keratinocyte differentiation. These results indicate that overexpression of PKCη only induces proliferation in physiologically proliferating cells and does not interfere with cell differentiation. Differences in the regulation of PKCn expression were also observed in vivo. Expression of PKCn was identified in actively proliferating skin as well as embryonic neuronal cells, whereas PKCn was not observed in the mature adult brain, where PKCn localized to the granule layer of the skin.

实施例11Example 11

PKCη和DNPKCη的过表达特别调控PKC的定位和细胞形态Overexpression of PKCη and DNPKCη specifically regulates the localization and cell morphology of PKC

为了进一步确证支持PKCη在角质化细胞的增殖状态或分化状态都具有积极作用的结果，通过研究感染对于增殖的和分化的角质化细胞的效果分析了激酶失活的显性失活腺病毒PKCη构建体的作用。如图12所示，在增殖状态和分化状态PKCη和DNPKCη的腺病毒感染都是高效的。正如所预测的那样，在增殖的角质化细胞中DNPKCη诱导角质化细胞的分化，同时使细胞形态发生巨大变化包括细胞变得扁平，细胞和细胞之间的边界消失，正与Ca²⁺诱导的分化伴随的形态改变类似(图12A-B)。而且，这些变化伴随着角质化细胞增殖的停止(图11)和分化标记包括角蛋白1，角蛋白10，兜甲蛋白(loricrin)和纤维聚合蛋白(filaggrin)的显著诱导，其量增加到与体内正常皮肤中类似的水平(图13A-B)。同时，随着分化程序的开始，DNPKCη的过表达不破坏Ca²⁺诱导的分化。这些结果表明PKCη和DNPKCη可用于在生理条件下有差别地调控角质化细胞的增殖和分化。To further corroborate the results supporting the positive role of PKCη in both the proliferative and differentiated states of keratinocytes, a kinase-inactive dominant negative adenoviral PKCη construct was analyzed by studying the effect of infection on proliferating and differentiating keratinocytes. the role of the body. As shown in FIG. 12, the adenovirus infection of PKCn and DNPKCn in both the proliferative state and the differentiation state was highly efficient. As predicted, DNPKCη induced differentiation of keratinocytes in proliferating keratinocytes, accompanied by dramatic changes in cell morphology including flattening of cells and disappearance of cell-to-cell boundaries, in parallel with Ca²⁺ -induced Morphological changes accompanying differentiation were similar (Fig. 12A-B). Moreover, these changes were accompanied by a cessation of keratinocyte proliferation (Fig. Similar levels in normal skin in vivo (Fig. 13A-B). At the same time, overexpression of DNPKCη did not disrupt Ca²⁺ -induced differentiation following the initiation of the differentiation program. These results suggest that PKCη and DNPKCη can be used to differentially regulate keratinocyte proliferation and differentiation under physiological conditions.

实施例12Example 12

体内实验in vivo experiment

为了检测PKCη在体内有差别地调控细胞增殖和分化的能力，评价了PKCη诱导裸鼠背部完全切开的创伤(full incisional wounds)愈合的能力。用对照β-gal腺病毒验证了角质化细胞表达外源重组蛋白的能力。如图14所示，感染后两周，在体外角质化细胞和体内皮肤都持续有β-gal表达。有趣的是，当用对照，PKCα和PKCη腺病毒构建体局部感染后检查小鼠的创伤愈合过程的时候，只有PKCη早在局部感染后四天就诱导肉芽组织形成。这还包括肌肉，脂肪和真皮层的有组织的形成。同时在对照和PKCα感染的皮肤中，没有观察到致密的肉芽组织，没有观察到创伤闭合(图14)。因此，PKCη可以视为是在诱导创伤愈合过程中调控皮肤增殖和分化的一个主要候选物。To test the ability of PKCη to differentially regulate cell proliferation and differentiation in vivo, the ability of PKCη to induce healing of full incisional wounds on the back of nude mice was evaluated. The ability of keratinocytes to express exogenous recombinant proteins was verified with a control β-gal adenovirus. As shown in Figure 14, β-gal expression persisted in both keratinocytes in vitro and skin in vivo two weeks after infection. Interestingly, when the wound healing process in mice was examined after topical infection with control, PKCα and PKCη adenoviral constructs, only PKCη induced granulation tissue formation as early as four days after topical infection. This also includes the organized formation of muscle, fat and dermal layers. Also in the control and PKCα-infected skin, no dense granulation tissue was observed and no wound closure was observed ( FIG. 14 ). Therefore, PKCη can be considered as a prime candidate for the regulation of skin proliferation and differentiation during the induction of wound healing.

实施例13Example 13

胰岛素在增殖的角质化细胞中特异诱导PKCδ的迁移Insulin specifically induces the migration of PKCδ in proliferating keratinocytes

已发现皮肤中表达的两种PKC同种型影响角质化细胞的增殖：PKCη和PKCδ。为了尝试和鉴别激活特定的调节皮肤增殖的PKC同种型的内源因子，评价了几种已知促进角质化细胞增殖的生长因子以生长依赖的方式激活特定PKC同种型的能力，包括：EGF，KGF，胰岛素，PDGF和IGF1。PKC同种型α，δ，ε，η和ζ在皮肤中表达。由于PKC同种型的激活与它们向膜组分的迁移有关，因此检查了这些生长因子对于不同PKC同种型从细胞溶质向膜迁移的作用。如图15所示，早在刺激后5分钟，胰岛素就特异诱导PKCδ从细胞质向膜组分迁移。胰岛素刺激后PKCδ在膜上的表达维持数小时。相反，IGF1减少了PKCδ在膜上的表达，而增加了其在细胞质组分中表达的相对水平。没有其它生长因子显著影响PKCδ的迁移和定位。其它PKC同种型在被任何生长因子包括IGF1和胰岛素刺激以后都没有观察到分布上的变化。Two isoforms of PKC expressed in the skin have been found to affect the proliferation of keratinocytes: PKCη and PKCδ. To try and identify endogenous factors that activate specific PKC isoforms that regulate skin proliferation, several growth factors known to promote keratinocyte proliferation were evaluated for their ability to activate specific PKC isoforms in a growth-dependent manner, including: EGF, KGF, insulin, PDGF and IGF1. PKC isoforms α, δ, ε, η and ζ are expressed in the skin. Since the activation of PKC isoforms is associated with their migration to membrane components, the effect of these growth factors on the migration of different PKC isoforms from the cytosol to the membrane was examined. As shown in Figure 15, insulin specifically induced the migration of PKCδ from the cytoplasm to the membrane fraction as early as 5 minutes after stimulation. Membrane expression of PKCδ is maintained for several hours after insulin stimulation. In contrast, IGF1 decreased the expression of PKCδ on the membrane while increasing its relative level in the cytoplasmic fraction. No other growth factors significantly affected the migration and localization of PKCδ. No change in distribution was observed for other PKC isoforms following stimulation by any growth factor, including IGF1 and insulin.

实施例14Example 14

胰岛素在增殖的角质化细胞中特异诱导PKCδ的活化Insulin specifically induces PKCδ activation in proliferating keratinocytes

为了确定PKCδ的迁移是否足以使其活化，测定了来自胰岛素和IGF1处理的角质化细胞中细胞质和膜组分的PKC免疫沉淀物的激酶活性。如图16所示，胰岛素而不是IGF1增加膜组分中PKCδ的活性。在细胞质组分中没有观察到PKCα活性的提高。胰岛素诱导的活化是对PKCδ特异性的，在胰岛素刺激后长达30分钟都没有观察到PKCα，ε，η或ζ的活化。总而言之，这些结果表明胰岛素而不是IGF1选择性刺激PKCδ的活化。To determine whether migration of PKCδ is sufficient for its activation, the kinase activity of PKC immunoprecipitates from cytoplasmic and membrane fractions in insulin- and IGF1-treated keratinocytes was assayed. As shown in Figure 16, insulin but not IGF1 increased the activity of PKCδ in the membrane fraction. No increase in PKCα activity was observed in the cytoplasmic fraction. Insulin-induced activation was specific for PKCδ, and no activation of PKCα, ε, η, or ζ was observed up to 30 minutes after insulin stimulation. Altogether, these results suggest that insulin, but not IGF1, selectively stimulates the activation of PKCδ.

实施例15Example 15

胰岛素和IGF1对于角质化细胞增殖具有加和作用Insulin and IGF1 have additive effects on keratinocyte proliferation

为了分析PKCδ的特异性活化是否意味着在角质化细胞中特定的胰岛素诱导的促有丝分裂途径，通过研究胰岛素和IGF1诱导角质化细胞增殖的能力检查了它们的促有丝分裂作用，通过胸苷掺入检测。如图17A所示，胰岛素和IGF1以剂量依赖的方式刺激胸苷掺入，分别在10^-7和10^-8M的时候达到最大诱导值。在每个浓度，IGF1的最大刺激值都比胰岛素高。有趣的是，在所有浓度，当两种激素一起使用的时候，具有加和的促有丝分裂作用(图17B)。这些结果表明胰岛素通过一种和IGF1诱导的角质化细胞增殖无关的不同途径调控角质化细胞的增殖。To analyze whether the specific activation of PKCδ implies a specific insulin-induced mitogenic pathway in keratinocytes, the mitogenic effects of insulin and IGF1 were examined by studying their ability to induce keratinocyte proliferation, detected by thymidine incorporation . As shown in Figure 17A, insulin and IGF1 stimulated thymidine incorporation in a dose-dependent manner, reaching maximum induction at 10^-7 and 10^-8 M, respectively. At each concentration, the maximal stimulation of IGF1 was higher than that of insulin. Interestingly, at all concentrations, the two hormones had additive mitogenic effects when used together (Fig. 17B). These results suggest that insulin regulates keratinocyte proliferation through a different pathway independent of IGF1-induced keratinocyte proliferation.

实施例16Example 16

胰岛素诱导的PKCδ活化和胰岛素诱导的角质化细胞增殖之间的关系Relationship Between Insulin-Induced PKCδ Activation and Insulin-Induced Keratinocyte Proliferation

为了直接研究胰岛素诱导的PKCδ活化和胰岛素诱导的角质化细胞增殖之间的关系，用重组PKC腺病毒构建体过表达野生型PKCδ(WTPKCδ)以及PKC的激酶失活的显性失活突变体，其破坏了内源PKCδ活性(DN PKCδ)。检查了WT PKCδ和DN PKCδ的过表达对于胰岛素诱导的角质化细胞增殖的作用。两个构建体以及PKCα构建体都能在角质化细胞中高效表达(图18A)。而且，用PKCδ和PKCα感染都可以比对照水平高出几倍地诱导同种型特异性的PKC活性(图18B)。正如所料，DN PKCδ的过表达不诱导PKC活性。如图19A所示，未转染细胞用胰岛素处理或不用胰岛素处理的WT PKCδ的过表达增加胸苷掺入到大约相同的水平，是未处理的细胞，或是用PKCα转导的细胞的二至三倍。而且，向已经过表达WT PKCδ的细胞中加入胰岛素不会引起胸苷掺入的任何额外增加。IGF1在未感染的细胞和过表达WT PKCδ和PKCα的细胞中类似地增加胸苷吸收(图19A)。进一步通过破坏PKCδ的活性证明PKCδ直接参与胰岛素诱导的增殖。如图19B所示，在过表达显性失活PKCδ的细胞中基础胸苷掺入轻微地，但显著地(significantly)，低于未感染细胞。DN PKCδ的过表达完全消除了胰岛素诱导的增殖，但是不影响IGF-1诱导的增殖。而且，胰岛素和IGF1的加和效果减至IGF1单独的水平。To directly study the relationship between insulin-induced PKCδ activation and insulin-induced keratinocyte proliferation, recombinant PKC adenoviral constructs were used to overexpress wild-type PKCδ (WTPKCδ) as well as a kinase-inactive dominant-negative mutant of PKC, It disrupts endogenous PKCδ activity (DN PKCδ). The effect of overexpression of WT PKCδ and DN PKCδ on insulin-induced keratinocyte proliferation was examined. Both constructs, as well as the PKCα construct, were highly expressed in keratinocytes (Fig. 18A). Moreover, infection with both PKCδ and PKCα induced isoform-specific PKC activity several-fold above control levels (Fig. 18B). As expected, overexpression of DN PKCδ did not induce PKC activity. As shown in Figure 19A, overexpression of WT PKCδ in untransfected cells treated with or without insulin treatment increased thymidine incorporation to approximately the same level as untreated cells, or the equivalent of cells transduced with PKCα. to three times. Moreover, addition of insulin to cells already overexpressing WT PKCδ did not cause any additional increase in thymidine incorporation. IGF1 similarly increased thymidine uptake in uninfected cells and cells overexpressing WT PKCδ and PKCα ( FIG. 19A ). It was further demonstrated that PKCδ was directly involved in insulin-induced proliferation by destroying the activity of PKCδ. As shown in Figure 19B, basal thymidine incorporation in cells overexpressing dominant negative PKCδ was slightly, but significantly, lower than in uninfected cells. Overexpression of DN PKCδ completely abolished insulin-induced proliferation but did not affect IGF-1-induced proliferation. Moreover, the additive effects of insulin and IGF1 were reduced to the level of IGF1 alone.

实施例17Example 17

PKCδ活化对胰岛素介导的途径的特异性Specificity of PKCδ activation for insulin-mediated pathways

通过研究PKCδ和DN PKCδ对于针对多种生长因子包括IGF1，EGF，KGF，ECGF和PDGF的促有丝分裂反应的作用来分析PKCδ活化对胰岛素介导的途径的特异性。如图20所示，DN PKCδ的过表达选择性地消除胰岛素诱导的增殖作用，但是不阻断所检测的任何其它生长因子的作用。但是，PKCδ的过表达模拟胰岛素诱导的增殖但不影响IGF1诱导的增殖。EGF和KGF的刺激诱导的增殖增加(图21)。这些数据表明胰岛素对PKCδ的活化通过涉及PKCδ的途径介导角质化细胞的增殖，这个途径位于EGF和KGF，两种主要的已知能调控角质化细胞增殖的生长因子的信号传导途径的上游。总的来说，发现胰岛素是PKCδ活性的特异性调节剂，其可能是在调控胰岛素，EGF和KGF诱导的角质化细胞增殖中的特异性候选物。The specificity of PKCδ activation to insulin-mediated pathways was analyzed by studying the role of PKCδ and DN PKCδ on mitogenic responses to multiple growth factors including IGF1, EGF, KGF, ECGF and PDGF. As shown in Figure 20, overexpression of DN PKCδ selectively abrogated insulin-induced proliferative effects, but did not block the effects of any of the other growth factors tested. However, overexpression of PKCδ mimics insulin-induced proliferation but does not affect IGF1-induced proliferation. Stimulation of EGF and KGF induced increased proliferation (Fig. 21). These data suggest that insulin activation of PKCδ mediates keratinocyte proliferation through a pathway involving PKCδ upstream of the signaling pathways of EGF and KGF, two major growth factors known to regulate keratinocyte proliferation. Collectively, insulin was found to be a specific modulator of PKCδ activity, which may be a specific candidate in the regulation of insulin, EGF and KGF-induced keratinocyte proliferation.

实施例18Example 18

胰岛素诱导的PKCδ活性角质化细胞增殖是由STAT3的转录激活介导的Insulin-induced PKCδ-active keratinocyte proliferation is mediated by transcriptional activation of STAT3

进一步研究PKCδ在胰岛素信号传导中的作用并发现其涉及STAT3介导的转录激活的诱导。如图23所示，在原代角质化细胞中，PKCδ特别与STAT3相关。在胰岛素刺激后，PKCδ被活化，然后磷酸化并激活STAT3(图24)。而且，用药理学抑制剂(卡马拉素)破坏PKCδ的活性抑制STAT3的活化以及其核定位。进一步，如图25所示，STAT3的过表达诱导与胰岛素诱导的和PKCδ过表达诱导的类似的增殖，过表达显性失活PKCδ突变体破坏PKCδ活性，破坏了STAT3诱导角质化细胞增殖的能力。这些结果总体表明胰岛素和PKCδ在与角质化细胞增殖相关的转录激活中发挥作用。The role of PKCδ in insulin signaling was further investigated and found to be involved in the induction of STAT3-mediated transcriptional activation. As shown in Figure 23, in primary keratinocytes, PKCδ is specifically associated with STAT3. After insulin stimulation, PKCδ is activated, which then phosphorylates and activates STAT3 (Fig. 24). Furthermore, disruption of PKCδ activity with a pharmacological inhibitor (kamaratin) inhibited STAT3 activation and its nuclear localization. Further, as shown in Figure 25, overexpression of STAT3 induces proliferation similar to that induced by insulin and overexpression of PKCδ, and overexpression of a dominant negative PKCδ mutant disrupts PKCδ activity and destroys the ability of STAT3 to induce keratinocyte proliferation . These results collectively suggest that insulin and PKCδ play a role in transcriptional activation associated with keratinocyte proliferation.

实施例19Example 19

PKCδ和PKCζ对于体内创伤愈合过程是必需的PKCδ and PKCζ are essential for wound healing processes in vivo

用同种型特异性PKC无效小鼠(PKC null mice)验证PKC同种型在体内创伤愈合过程中的重要性。如图22A-B所示，当在PKCδ，PKCζ，PKCα无效小鼠(基因敲除，KO)和它们的野生型同窝出生鼠的背部造成全厚度创伤的时候，在PKCδ和PKCζ无效小鼠中观察到创伤愈合延迟，而PKCα无效小鼠没有。这个结果表明甚至是在没有糖尿病背景下，特定的PKC同种型对于皮肤的创伤愈合过程也是必需的。Validation of the importance of PKC isoforms during wound healing in vivo using isoform-specific PKC null mice. As shown in Figure 22A-B, when full-thickness wounds were inflicted on the backs of PKCδ, PKCζ, PKCα null mice (knockout, KO) and their wild-type littermates, the PKCδ and PKCζ null mice Delayed wound healing was observed in , but not in PKCα-null mice. This result suggests that specific PKC isoforms are essential for the wound-healing process of the skin even in the absence of diabetes.

实施例20Example 20

用于体内创伤愈合的胰岛素的单次应用与多次应用Single versus multiple applications of insulin for wound healing in vivo

通过切割在8-10周龄C57BL小鼠背部造成创伤，然后进行如下处理：(i)在7天中每天施用胰岛素0.1μM；(ii)在7天中每天施用胰岛素1μM；(iii)在7天中每天施用胰岛素10μM；(iv)在致创后4天一次性施用胰岛素1μM；以及(v)在7天中每天施用载体(PBS)对照。所有小鼠都在致创后七天处死，测量它们的张开的创伤的面积。如图26所示，以1μM的浓度每天用胰岛素处理显著地比用更低(0.1μM)或更高(10μM)浓度的胰岛素每天处理更有效。令人惊讶地是，以1μM的浓度单次施用胰岛素比用相同浓度的胰岛素每天施用重复七天处理明显更有效。The back of 8-10-week-old C57BL mice was wounded by cutting, and then treated as follows: (i) insulin 0.1 μM per day for 7 days; (ii)insulin 1 μM per day for 7 days; (iii)insulin 1 μM per day for 7 days; Insulin 10 [mu]M was administered daily for days; (iv) insulin 1 [mu]M was administered as a single dose 4 days after wounding; and (v) vehicle (PBS) control was administered daily for 7 days. All mice were sacrificed seven days after wounding and the area of their open wounds was measured. As shown in Figure 26, daily insulin treatment at a concentration of 1 μM was significantly more effective than daily treatment with lower (0.1 μM) or higher (10 μM) concentrations of insulin. Surprisingly, a single administration of insulin at a concentration of 1 [mu]M was significantly more effective than repeated seven-day treatment with daily administration of the same concentration of insulin.

由于观察到的创伤被疤痕组织覆盖，很难正确评价创伤的实际闭合和重建的表皮的形成。因此用组织学参数来确定胰岛素对于创伤组织的表皮和真皮的闭合的效果。创伤的表皮闭合是通过用角蛋白14抗体(K14，Babco-Convance，Richmond，CA，USA)对创伤切片(sections)染色来确定的，它能够突出显示创伤裂口上基底细胞的形成。如果真皮创伤的两边在x100倍放大的光学显微镜下在一个视野中都能看见，则创伤的真皮闭合视为阳性。Since the observed wound is covered by scar tissue, it is difficult to properly assess the actual closure of the wound and the formation of a reconstructed epidermis. Histological parameters were therefore used to determine the effect of insulin on the closure of the epidermis and dermis of wounded tissue. Epidermal closure of wounds was determined by staining wound sections with an antibody to keratin 14 (K14, Babco-Convance, Richmond, CA, USA), which highlights the formation of basal cells on wound clefts. Dermal closure of the wound was considered positive if both sides of the dermal wound were visible in one field under a light microscope at x100 magnification.

如图27所示，所有胰岛素处理都能有效促进表皮和真皮闭合。与图26所示的结果类似，每天用1μM浓度的胰岛素处理显著比用0.1μM或10μM浓度的胰岛素每天处理更有效。此外，用1μM浓度的胰岛素单次施用比用相同浓度的胰岛素每天施用重复七天处理明显更有效。As shown in Figure 27, all insulin treatments were effective in promoting epidermal and dermal closure. Similar to the results shown in Figure 26, daily treatment with insulin at a concentration of 1 μM was significantly more effective than daily treatment with insulin at a concentration of 0.1 μM or 10 μM. Furthermore, a single administration of insulin at a concentration of 1 μM was significantly more effective than repeated seven-day treatment with daily administration of the same concentration of insulin.

因此，由形态学和组织学参数得到的这些结果显然证明胰岛素对于体内创伤愈合的治疗效果。这些结果令人惊讶地表明确定胰岛素施用的最佳数目和／或频率是正确治疗创伤的关键步骤。Therefore, these results obtained from morphological and histological parameters clearly demonstrate the therapeutic effect of insulin on wound healing in vivo. These results surprisingly indicate that determining the optimal number and/or frequency of insulin administration is a critical step in the proper treatment of wounds.

实施例21Example 21

组合使用胰岛素和血小板衍生生长因子(PDGF-BB)用于体内创伤愈合Combined use of insulin and platelet-derived growth factor (PDGF-BB) for wound healing in vivo

通过切割在8-10周龄C57BL小鼠背部造成创伤，致创4天后进行如下处理：(i)载体(PBS)对照；(ii)胰岛素1μM；(iii)PDGF-BB10μM(R&D Systems，Minneapolis，USA)；和(iv)胰岛素1μM+PDGF-BB10μM。处理后三天处死所有小鼠，对处理的创伤从组织学上分析表皮和真皮闭合，如上述实施例20中所述。Injury was made on the back of 8-10 week-old C57BL mice by cutting, and the following treatments were carried out 4 days after wounding: (i) vehicle (PBS) control; (ii)insulin 1 μM; (iii) PDGF-BB 10 μM (R&D Systems, Minneapolis, USA); and (iv)insulin 1 μM + PDGF-BB 10 μM. All mice were sacrificed three days after treatment, and treated wounds were histologically analyzed for epidermal and dermal closure, as described in Example 20 above.

如图28所示，单独用胰岛素或PDGF-BB处理对于表皮闭合(比对照高30-40％)和真皮闭合(比对照高10-20％)都是部分有效。但是用胰岛素和PDGF-BB组合处理产生了明显更高的表皮闭合(大约比对照高80％)以及真皮闭合(大约60％)。因此，这些结果表明组合使用胰岛素和PDGF-BB能以协同作用的方式影响创伤愈合。这些结果还表明可将胰岛素和其它生长因子或转化因子例如EGF，TGFβ，KGF组合使用用于创伤的治疗性处理。As shown in Figure 28, treatment with either insulin or PDGF-BB alone was partially effective in both epidermal closure (30-40% greater than control) and dermal closure (10-20% greater than control). However, combined treatment with insulin and PDGF-BB produced significantly higher epidermal closure (approximately 80% higher than control) as well as dermal closure (approximately 60%). Thus, these results suggest that the combined use of insulin and PDGF-BB can affect wound healing in a synergistic manner. These results also suggest that insulin can be used in combination with other growth factors or transforming factors such as EGF, TGF[beta], KGF for the therapeutic management of wounds.

实施例22Example 22

组合使用胰岛素和PKCα抑制剂用于体内创伤愈合Combined use of insulin and PKCα inhibitors for wound healing in vivo

通过切割在8-10周龄C57BL小鼠背部造成创伤，用载体(PBS)对照或0.67μM胰岛素(HO／01；Humulin，Eli Lilly，USA)和PKCα抑制剂(HO／02；十四酰化的PKCα伪底物；Calibiochem，San Diego，CA，USA)的组合每天处理连续7天。致创后7天处死所有小鼠，对被处理的创伤分析其创伤闭合，表皮闭合，真皮闭合，和表皮细胞的空间分化。测定张开的创伤区域以确定创伤闭合。如果真皮创伤的两边在x100倍放大的光学显微镜下在一个视野中都能看见，则创伤的真皮闭合视为阳性。用突出显示创伤裂口上基底细胞的形成的k14抗体对创伤切片进行染色来确定创伤的表皮闭合。表皮细胞的空间分化通过用突出显示新形成的表皮细胞的k1抗体对创伤切片进行染色来确定。Wounding was done on the back of 8–10 week-old C57BL mice by excision, treated with vehicle (PBS) control or 0.67 μM insulin (HO/01; Humulin, Eli Lilly, USA) and a PKCα inhibitor (HO/02; myristylated PKCα pseudo-substrates; Calibiochem, San Diego, CA, USA) were treated daily for 7 consecutive days. All mice were sacrificed 7 days after wounding, and the treated wounds were analyzed for wound closure, epidermal closure, dermal closure, and spatial differentiation of epidermal cells. The open wound area is measured to determine wound closure. Dermal closure of the wound was considered positive if both sides of the dermal wound were visible in one field under a light microscope at x100 magnification. Epidermal closure of the wound was determined by staining wound sections with a k14 antibody that highlights the formation of basal cells on the wound breach. Spatial differentiation of epidermal cells was determined by staining wound sections with a K1 antibody that highlights newly formed epidermal cells.

如图28-32所示，组合使用胰岛素和(HO／01)和PKCα抑制剂(HO／02)显著能促进创伤闭合(图29A-B)，真皮闭合(图30)，表皮闭合(图31)，和表皮细胞的空间分化(图32)。如图33所示，与载体对照相比，组合使用胰岛素HO／01和PKCα抑制剂HO／02进行处理可分别将创伤表皮闭合从大约15％提高到70％，将真皮闭合从大约15％提高到50％，将表皮细胞的空间分化从大约15％提高到50％。As shown in Figures 28-32, the combination of insulin and (HO/01) and PKCα inhibitor (HO/02) can significantly promote wound closure (Figure 29A-B), dermal closure (Figure 30), epidermal closure (Figure 31 ), and spatial differentiation of epidermal cells (Figure 32). As shown in Figure 33, treatment with the combination of insulin HO/01 and the PKCα inhibitor HO/02 increased wound epidermal closure from approximately 15% to 70% and dermal closure from approximately 15%, respectively, compared to vehicle control to 50%, increasing the spatial differentiation of epidermal cells from approximately 15% to 50%.

因此，这些结果表明组合使用胰岛素和PKCα抑制剂对创伤进行治疗性处理有效促进表皮闭合，真皮闭合，表皮细胞的空间分化，和随后的创伤愈合。Thus, these results demonstrate that therapeutic wound treatment using a combination of insulin and a PKCα inhibitor is effective in promoting epidermal closure, dermal closure, spatial differentiation of epidermal cells, and subsequent wound healing.

实施例23Example 23

组合使用胰岛素和PKCα抑制剂可防止单独使用胰岛素处理引起的不良的副作用Combination of insulin and PKCα inhibitor prevents adverse side effects of insulin treatment alone

通过切割在8-10周龄C57BL小鼠背部造成创伤，用载体(PBS)对照或1μM胰岛素(Humulin，Eli Lilly，USA)或者1μM胰岛素和1μMPKCα伪底物(Calibiochem，San Diego，CA，UJSA)的混合物每天处理连续7天。致创后7天处死全部小鼠，对被处理的创伤从组织学上分析其表皮的增殖能力(PCNA)，血管生成，炎症，表皮细胞和创伤裂口的重建过程。Wounding was done on the back of 8-10 week old C57BL mice by dissection with vehicle (PBS) control or 1 μM insulin (Humulin, Eli Lilly, USA) or 1 μM insulin and 1 μM M PKCα pseudo-substrate (Calibiochem, San Diego, CA, UJSA) The mixture was treated daily for 7 consecutive days. All mice were sacrificed 7 days after wounding, and the treated wounds were analyzed histologically for the proliferative ability (PCNA), angiogenesis, inflammation, epidermal cells and the reconstruction process of wound gap.

如下述表1所示，与缓冲液对照相比，仅用胰岛素处理会在创伤部位引起异常血管生成的大幅增大(分别为60％和25％)。由于创伤愈合过程涉及快速增殖的表皮细胞，因此血管生成的增加也可能会增加引发癌症发展的危险。另一方面，当组合使用胰岛素和PKCα抑制剂的时候，在被处理的创伤部位没有观察到血管生成。As shown in Table 1 below, treatment with insulin alone caused a substantial increase in abnormal angiogenesis at the wound site (60% vs. 25%, respectively) compared to the buffer control. Because the wound healing process involves rapidly proliferating epidermal cells, increased angiogenesis may also increase the risk of cancer development. On the other hand, when insulin and PKCα inhibitors were used in combination, no angiogenesis was observed in the treated wound sites.

表1Table 1

单独使用胰岛素和组合使用胰岛素和PKCα抑制剂对创伤部位的血管生成的严重程度的影响Effect of insulin alone and in combination with insulin and PKCα inhibitors on the severity of angiogenesis at trauma sites

此外，仅用胰岛素进行处理会导致炎症增加，表皮细胞增生，表皮细胞棘层分化延迟和疤痕增加。当PKCα抑制剂和胰岛素组合在一起的时候没有观察到使用胰岛素单独进行处理导致的不良副作用。Furthermore, treatment with insulin alone resulted in increased inflammation, epidermal hyperplasia, delayed acanthotic differentiation of epidermal cells, and increased scarring. The adverse side effects of treatment with insulin alone were not observed when the PKCα inhibitor was combined with insulin.

实施例24Example 24

PKCα抑制剂减少创伤炎症PKCα inhibitors reduce wound inflammation

创伤处晚期的和严重的炎症反应可能会抑制愈合过程，因此防止这种炎症发生可促进创伤愈合过程。相应地，在下面的实验中检验了PKCα抑制剂和胰岛素对创伤炎症的作用。Late and severe inflammatory responses at the wound site may inhibit the healing process, thus preventing this inflammation from occurring may promote the wound healing process. Accordingly, the effects of PKCα inhibitors and insulin on wound inflammation were examined in the following experiments.

通过切割在C57BL小鼠背部造成创伤，每天进行如下处理连续7天：(i)PBS，对照；(ii)1μMPKCα抑制剂(十四酰化的伪底物；Calibiochem，USA)；(iii)1μM胰岛素(Eli Lilly，USA)；或1μM PKCα抑制剂和1μM胰岛素的混合物。致创后7天处死所有小鼠，在显微镜下观察被处理创伤的炎症。在创伤部位观察到的严重炎症的发生率总结在下述表1a中。Wounding was performed on the back of C57BL mice by cutting, and the following treatments were performed every day for 7 consecutive days: (i) PBS, control; (ii) 1 μM KCα inhibitor (myristylated pseudosubstrate; Calibiochem, USA); (iii) 1 μM Insulin (Eli Lilly, USA); or a mixture of 1 μM PKCα inhibitor and 1 μM insulin. All mice were sacrificed 7 days after wounding, and the inflammation of treated wounds was observed under a microscope. The incidence of severe inflammation observed at the wound site is summarized in Table 1a below.

表1aTable 1a

处理deal with创伤中严重炎症的发生率(％)Incidence of severe inflammation in trauma (%)PBS对照PBS control60.060.0PKCα抑制剂PKCα inhibitors40.040.0胰岛素insulin56.056.0PKCα抑制剂+胰岛素PKCα inhibitor + insulin50.050.0

结果表明与对照相比，给创伤施用PKCα抑制剂可使严重创伤炎症的发生率实质(33.3％)减少。在实验条件下单独使用胰岛素没有抗炎性效果。The results show that administration of a PKCα inhibitor to wounds substantially (33.3%) reduces the incidence of severe wound inflammation compared to controls. Insulin alone had no anti-inflammatory effect under the experimental conditions.

这些结果表明PKCα抑制剂可用于控制创伤的严重炎症的治疗。已证明的PKCα抑制剂的减少炎症的能力与它能促进表皮闭合，真皮闭合和表皮细胞的空间分化的能力(参见上述实施例22)，使得它可能成为创伤愈合的最有效的治疗剂。These results suggest that PKCα inhibitors may be useful in the treatment of severe inflammation in wounds. The demonstrated ability of a PKCα inhibitor to reduce inflammation combined with its ability to promote epidermal closure, dermal closure and spatial differentiation of epidermal cells (see Example 22 above) makes it potentially the most effective therapeutic agent for wound healing.

实施例25Example 25

调节真皮细胞中特定PKC同种型的表达和／或活性和给细胞施用不同试剂对加速体外创伤闭合的组合效果Combinatorial effect of modulating the expression and/or activity of specific PKC isoforms in dermal cells and administering different agents to the cells on accelerating wound closure in vitro

材料和方法：Materials and methods:

试剂：因子D(Adipsin)人，CalbioChem，California USA；重组TNFα小鼠，R&D Systems，Minneapolis USA；GW9662，Caymanchemical，USA；蛋白激酶Cα伪底物抑制剂，CalbioChem，CaliforniaUSA；蛋白激酶Cζ伪底物抑制剂，CalbioChem，California USA；蛋白激酶Cη伪底物抑制剂，CalbioChem，California USA；PDGF-BB，Cytolab，Israel；IL-6，Cytolab，Israel；KGF／FGF-7，Cytolab，Israel；IGF-1，Cytolab，Israel；TGFβ2，Cytolab，Israel；表皮生长因子(EGF)，小鼠，Chemicon international，CaliforniaUSA；PKCδRACK，AnaSpec，California USA；罗格列酮(Rosiglitazon)，CalbioChem.California USA；脂联素(Adiponectin)，MBL，Massachusetts USA and

TEVA.Israel。Reagents: Factor D (Adipsin) human, CalbioChem, California USA; recombinant TNFα mouse, R&D Systems, Minneapolis USA; GW9662, Caymanchemical, USA; protein kinase Cα pseudosubstrate inhibitor, CalbioChem, California USA; protein kinase Cζ pseudosubstrate Inhibitor, CalbioChem, California USA; Pseudo-substrate inhibitor of protein kinase Cη, CalbioChem, California USA; PDGF-BB, Cytolab, Israel; IL-6, Cytolab, Israel; KGF/FGF-7, Cytolab, Israel; IGF- 1, Cytolab, Israel; TGFβ2, Cytolab, Israel; epidermal growth factor (EGF), mouse, Chemicon international, California USA; PKCδRACK, AnaSpec, California USA; Rosiglitazon, CalbioChem.California USA; adiponectin (Adiponectin), MBL, Massachusetts USA and

TEVA. Israel.

体外创伤闭合检测：在皮氏培养皿(5cm i.d.)中将角质化细胞和成纤维细胞(真皮细胞)培养五天，然后用200μl的移液管头在每个培养皿中形成人工的交叉划痕。将培养的细胞用能调节特定PKC同种型的表达和／或活性的腺病毒构建体感染。相应地，用野生型(WT)PKC腺病毒构建体活化特定的PKC，用显性失活(DN)PKC腺病毒构建体抑制特定的PKC。向培养的细胞中进一步加入下述试剂中的一种：胰岛素(6.7x10^-7M)，脂联素(每培养皿1μg)，adipsin(2μg／ml)，IL-6(每培养皿1μg)，GW9662(每培养皿1μg)，KGF(每培养皿1μg)，TNFα(12μg／ml)，TGFβ，rosiglitazone，SRC抑制剂，PKCδRACK(10^-7M)和PKCα伪底物抑制肽(10⁷M)。在处理后24-48小时确定得到的创伤闭合水平，用从0(不闭合)到10(完全闭合)的指数值表示。In vitro wound closure assay: Culture keratinocytes and fibroblasts (dermal cells) in petri dishes (5 cm id) for five days, then use a 200 μl pipette tip to form an artificial cross-hatch in each dish mark. Cultured cells are infected with an adenoviral construct capable of modulating the expression and/or activity of a particular PKC isoform. Accordingly, specific PKCs were activated with wild-type (WT) PKC adenoviral constructs, and specific PKCs were inhibited with dominant negative (DN) PKC adenoviral constructs. Add one of the following reagents to the cultured cells: insulin (6.7x10^-7 M), adiponectin (1 μg per culture dish), adipsin (2 μg/ml), IL-6 (1 μg per culture dish) , GW9662 (1 μg per dish), KGF (1 μg per dish), TNFα (12 μg/ml), TGFβ, rosiglitazone, SRC inhibitor, PKCδRACK (10^-7 M) and PKCα pseudosubstrate inhibitory peptide (10⁷ M ). The resulting level of wound closure was determined 24-48 hours after treatment, expressed as an index value from 0 (no closure) to 10 (complete closure).

结果：result:

组合治疗对体外成纤维细胞创伤闭合的作用总结于下表2a-b和3a-b。结果表明当配合adipsin或胰岛素对细胞给药的时候，成纤维细胞中PKCα表达和／或活性的抑制显著促进创伤闭合(创伤闭合的指数值分别为10和8)。在成纤维细胞中PKCα的抑制与PKCη的抑制，PKCε的抑制，PKCδ的活化，或PKCζ的活化相组合也加速创伤闭合(创伤闭合的指数值分别为9，9，9和7；图34A-E)。此外，在成纤维细胞中PKCζ的抑制与KFG对细胞的给药相组合促进创伤愈合(创伤愈合指数值为7；图36)。进一步另外，成纤维细胞中PKCβ的抑制与胰岛素，IL-6，KGF或GW9662的给药相组合加速创伤闭合(创伤闭合指数值分别为8，7，9和8；图38A-E)。The effects of combination therapy on fibroblast wound closure in vitro are summarized in Tables 2a-b and 3a-b below. The results showed that when the cells were administered with adipsin or insulin, the inhibition of PKCα expression and/or activity in fibroblasts significantly promoted wound closure (the index values of wound closure were 10 and 8, respectively). Inhibition of PKCα in fibroblasts combined with inhibition of PKCη, inhibition of PKCε, activation of PKCδ, or activation of PKCζ also accelerated wound closure (index values for wound closure were 9, 9, 9, and 7, respectively; FIG. 34A- E). Furthermore, inhibition of PKCζ in fibroblasts in combination with KFG administration to the cells promoted wound healing (WoundHealing Index value 7; Figure 36). Further additionally, inhibition of PKCβ in fibroblasts combined with administration of insulin, IL-6, KGF or GW9662 accelerated wound closure (wound closure index values of 8, 7, 9 and 8, respectively; Figure 38A-E).

表2aTable 2a

组合处理对体外创伤成纤维细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Fibroblasts in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

ND=未测定ND = not determined

表2bTable 2b

组合处理对体外创伤成纤维细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Fibroblasts in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

表3aTable 3a

组合处理对体外创伤成纤维细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Fibroblasts in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

表3bTable 3b

组合处理对体外创伤成纤维细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Fibroblasts in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

组合处理对于体外角质化细胞创伤闭合的效果总结于下表4a-b和5a-b中。结果显示当配合KGF，IL-6，TNFα或PKCδRACK肽对细胞给药的时候，角质化细胞中PKCα表达和／或活性的抑制显著促进创伤闭合(创伤闭合的指数值分别为6，8，10和8；图35A-C和G)。角质化细胞中PKCα的抑制与细胞中PKCη，PKCε，或PKCζ的刺激相组合也增加创伤闭合(创伤闭合的指数值分别为10，9和6；图35A，D-F和H)。此外，角质化细胞中PKCζ的抑制与IL-6，TNF-α或脂联素对细胞的给药相组合促进创伤闭合(创伤闭合指数值分别为9，9和7；图37A-D)。进一步另外，在角质化细胞中提高PKCδ的活性和／或表达与细胞中PKCε的激活，PKCζ的激活，或PKCα的抑制相组合，或者adipsin向细胞的给药，加速创伤闭合(创伤闭合指数值分别为7，8，8和8；图39A-E)。The effect of the combination treatment on keratinocyte wound closure in vitro is summarized in Tables 4a-b and 5a-b below. The results showed that when KGF, IL-6, TNFα or PKCδRACK peptide was administered to the cells, the inhibition of PKCα expression and/or activity in keratinocytes significantly promoted wound closure (the index values of wound closure were 6, 8, 10, respectively). and 8; Figures 35A-C and G). Inhibition of PKCα in keratinocytes combined with stimulation of PKCη, PKCε, or PKCζ in cells also increased wound closure (index values for wound closure were 10, 9, and 6, respectively; Figure 35A, D-F, and H). Furthermore, inhibition of PKCζ in keratinocytes in combination with administration of IL-6, TNF-α or adiponectin to cells promoted wound closure (Wound Closure Index values of 9, 9 and 7, respectively; Figure 37A-D). Further additionally, increasing PKCδ activity and/or expression in keratinocytes in combination with activation of PKCε, activation of PKCζ, or inhibition of PKCα in cells, or administration of adipsin to cells, accelerated wound closure (Wound Closure Index values 7, 8, 8 and 8, respectively; Figure 39A-E).

表4aTable 4a

组合处理对体外创伤角质化细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Keratinocytes in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

ND=未测定ND = not determined

表4bTable 4b

组合处理对体外创伤角质化细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Keratinocytes in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

ND=未测定ND = not determined

表5aTable 5a

组合处理对体外创伤角质化细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Keratinocytes in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

ND=未测定ND = not determined

表5bTable 5b

组合处理对体外创伤角质化细胞闭合的效果¹Effects of Combination Treatment on Closure of Wounded Keratinocytes in Vitro¹

¹闭合的数值是0(不闭合)到10(完全闭合)¹ The value of closure is 0 (not closed) to 10 (completely closed)

ND=未测定ND = not determined

因此，该结果表明当联合生长因子例如IL-6，KGF，TNFα，激素例如胰岛素，脂肪细胞激素例如adipsin或脂联素，PKCδRACK和／或GW9662对细胞的给药时，调节创伤部位的真皮细胞和表皮细胞中特定PKC同种型的表达和／或活性显著加速创伤闭合。Thus, the results indicate that regulation of dermal cells at the site of wounds when administered to the cells in combination with growth factors such as IL-6, KGF, TNFα, hormones such as insulin, adipocyte hormones such as adipsin or adiponectin, PKCδRACK and/or GW9662 Expression and/or activity of specific PKC isoforms in epidermal cells significantly accelerated wound closure.

实施例26Example 26

体外和体内施用共聚物-1用于创伤愈合In vitro and in vivo administration of copolymer-1 for wound healing

材料和方法：Materials and methods:

共聚物-1：共聚物-1(Glatiramer acetate)是药物(Teva，Israel)的活性成分，在临床上用于治疗多发性硬化。共聚物-1是髓鞘碱性蛋白(MBP)的合成多肽类似物，MBP是髓鞘的天然成分。从化学上来分析，共聚物-1是L-谷氨酸和L-丙氨酸，L-赖氨酸和L-酪氨酸的聚合物的乙酸盐。它的结构式是：(Glu，Ala，Lys，Tyr)x.X CH3COOH(C5H9NO4·C3H7NO2·C6H14N2O2·C9H11NO3)x·x C2H4O2。glatiramer acetate的平均分子量是4,700-11,000道尔顿。它的合成是将这四种氨基酸进行化学聚合形成平均分子量为23,000道尔顿的产物(美国专利No.3,849,550)。Copolymer-1: Copolymer-1 (Glatiramer acetate) is a drug (Teva, Israel), clinically used in the treatment of multiple sclerosis. Copolymer-1 is a synthetic polypeptide analog of myelin basic protein (MBP), a natural component of myelin sheath. Chemically, Copolymer-1 is an acetate salt of a polymer of L-glutamic acid and L-alanine, L-lysine and L-tyrosine. Its structural formula is: (Glu, Ala, Lys, Tyr)xX CH3COOH (C5H9NO4·C3H7NO2·C6H14N2O2·C9H11NO3)x·x C2H4O2. The average molecular weight of glatiramer acetate is 4,700-11,000 Daltons. It is synthesized by chemically polymerizing these four amino acids to form a product with an average molecular weight of 23,000 Daltons (US Patent No. 3,849,550).

体外检测：基本按照上述实施例24中所述进行检测。向培养的角质化细胞中加入共聚物-1，浓度为每个培养皿55μg，单独加入或与PKCα伪底物(1μM)和／或胰岛素(1μM)一起加入。处理后48小时确定得到的创伤闭合水平，采用从0(不闭合)到10(完全闭合)的指数值。In vitro detection: detection was basically performed as described in Example 24 above. Copolymer-1 was added to cultured keratinocytes at a concentration of 55 μg per dish, alone or together with PKCα pseudosubstrate (1 μM) and/or insulin (1 μM). The resulting level of wound closure was determined 48 hours after treatment using an index value from 0 (no closure) to 10 (complete closure).

体内检测：通过切割在C57BL小鼠背部造成创伤(20mm)，致创4天后进行如下处理：(i)载体(PBS)对照；(ii)共聚物-1(55μg／ml)；(iii)共聚物-1(55μg／ml)和胰岛素(1μM)的混合物；(iv)PKCα伪底物抑制肽(1μM)和胰岛素(1μM)的混合物；以及(v)共聚物-1(55μg／ml)，PKCα伪底物抑制肽(1μM)和胰岛素(1μM)的混合物。从形态学上对创伤评估其(i)创伤闭合，(ii)痂的形成和(iii)创伤的出血／渗液。In vivo testing: Wounds (20 mm) were made on the back of C57BL mice by cutting, and the following treatments were performed 4 days after the wounding: (i) vehicle (PBS) control; (ii) copolymer-1 (55 μg/ml); (iii) copolymerization mixture of substance-1 (55 μg/ml) and insulin (1 μM); (iv) mixture of PKCα pseudo-substrate inhibitory peptide (1 μM) and insulin (1 μM); and (v) copolymer-1 (55 μg/ml), Mixture of PKCα pseudo-substrate inhibitory peptide (1 μM) and insulin (1 μM). Wounds were assessed morphologically for (i) wound closure, (ii) scab formation and (iii) wound bleeding/exudate.

结果result

体外检测：给培养的角质化细胞施用共聚物-1促进体外创伤闭合，在指数值范围为0(不闭合)到10(完全闭合)的尺度中达到指数值为8。将共聚物-1与PKCα伪底物抑制肽组合，或与PKCα伪底物和胰岛素的混合物组合，获得相似的效果(创伤闭合指数值分别为8和9；图40A-F)。因此，这些结果表明共聚物-1本身能够显著加速体外创伤闭合。In Vitro Assays: Administration of Copolymer-1 to cultured keratinocytes promotes wound closure in vitro, reaching an index value of 8 on a scale of index values ranging from 0 (no closure) to 10 (complete closure). Combining Copolymer-1 with a PKCa pseudo-substrate inhibitory peptide, or with a mixture of a PKCa pseudo-substrate and insulin, achieved similar effects (wound closure index values of 8 and 9, respectively; Figure 40A-F). Thus, these results indicate that Copolymer-1 itself is capable of significantly accelerating wound closure in vitro.

体内检测：与未处理的对照相比，给切开的创伤单独施用共聚物-1，或与胰岛素和／或PKCα伪底物组合施用，能够显著减少创伤裂口区域并加速创伤中痂的形成。此外，所有用共聚物-1进行的处理都有效防止创伤部位出血和渗液。In Vivo Assays: Administration of Copolymer-1 alone or in combination with insulin and/or PKCα pseudo-substrates to incised wounds significantly reduced wound dehiscence area and accelerated scab formation in wounds compared to untreated controls. In addition, all treatments with copolymer-1 were effective in preventing bleeding and exudation at the wound site.

因此，这些结果表明给创伤部位单独施用有效量的共聚物-1或与胰岛素和／或PKCα伪底物抑制肽组合施用可以能够显著加速创伤愈合过程。Thus, these results indicate that administering to the wound site an effective amount of Copolymer-1 alone or in combination with insulin and/or PKCα pseudo-substrate inhibitory peptide can significantly accelerate the wound healing process.

实施例27Example 27

胸腺分泌的物质对于创伤愈合过程的影响Effects of Thymus Secreted Substances on Wound Healing Process

材料和方法：Materials and methods:

在正常的成年啮齿动物或STZ糖尿病小鼠的上背部(靠近脖颈处)切出创伤。处理后7或9天处死动物，从组织学对创伤分析创伤部位附近胸腺的存在，用上述实施例20中所述的染色程序分析创伤的表皮和真皮闭合。Wounds were excised in the upper back (near the neck) of normal adult rodents or STZ diabetic mice. Animals were sacrificed 7 or 9 days after treatment and the wounds were analyzed histologically for the presence of thymus adjacent to the wound site and for epidermal and dermal closure of the wounds using the staining procedure described in Example 20 above.

结果：result:

如图42A-H所示，在创伤裂口紧邻处胸腺的存在与创伤中加速的上皮化，组织的粒化和真皮收缩有关。这些观察结果表明胸腺分泌的物质可有效利于创伤的愈合过程。相应地，胸腺衍生的物质例如胸腺素，β胸腺素(例如胸腺素β4，胸腺素β10，胸腺素β9，胸腺素β12，胸腺素β14)，αthimosin(例如胸腺素α，1／zadaxin，前胸腺素α，parathymosinα)，胸腺肽，IGFI，IGFII，NGF，生长激素抑制素，甲状腺球蛋白，副甲状腺激素和／或胸腺激素肽(THP)可用于加速创伤愈合过程的治疗。As shown in Figures 42A-H, the presence of thymus in the immediate vicinity of the wound breach was associated with accelerated epithelialization, tissue granulation and dermal contraction in the wound. These observations suggest that substances secreted by the thymus may effectively facilitate the wound healing process. Correspondingly, thymus-derived substances such as thymosin, β thymosin (e.g. thymosin β4, thymosin β10, thymosin β9, thymosin β12, thymosin β14), αthimosin (e.g. thymosin α, 1/zadaxin, prothymosin Thymosin α, parathymosin α), thymosin, IGFI, IGFII, NGF, somatostatin, thyroglobulin, parathyroid hormone and/or thymosin peptide (THP) can be used in the treatment of accelerated wound healing process.

应当理解在分开的实施方案中为清楚起见描述的本发明的特定特征，也可以在单个实施方案中组合提供。相反地，在单个实施方案中为简洁起见描述的本发明的各个特征，也可以分开提供或者以任何合适的次组合提供。It is to be understood that certain features of the invention, which are, for clarity, described in separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

虽然本发明是结合特定的实施方案描述的，很明显对于本领域技术人员来说有多种替代方案，修改和变化都是显而易见地。相应地，本发明意在包括落在本发明的附加的权利要求的精神和范围中的所有替代方案，修改和变化。说明书中提到的所有出版物，专利，专利申请都将其全文引入说明书中作为参考，如同特别地和单独指出每一个出版物，专利，专利申请都被引入本文作为参考。此外，本申请中对任何参考文献的引用或标识不应当理解为承认这些文献可作为本发明的现有技术。While the invention has been described in conjunction with specific embodiments thereof, it is evident that various alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the invention is intended to embrace all alternatives, modifications and variations that fall within the spirit and scope of the appended claims of the invention. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference herein. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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42.Braiman，L.，Alt，A.，Kuroki,T.，Ohba，M.，Bak，A.，Tennenbaum,T.，and Sampson，S.R.Protein kinase Cdelta mediates insulin-induced glucose transportin primary cultures of rat skeletal muscle.Mol.Endocrinol.，13：2002-2012，1999.42. Braiman, L., Alt, A., Kuroki, T., Ohba, M., Bak, A., Tennenbaum, T., and Sampson, S.R. Protein kinase Cdelta mediates insulin-induced glucose transportin primary cultures of rat skeletal muscle. Mol. Endocrinol., 13: 2002-2012, 1999.

43.Braiman，L.，Sheffi-Friedman，L.，Bak,A.，Tennenbaum，T.，andSampson，S.R.Tyrosine phosphorylation of specific protein kinase C isoenzymesparticipates in insulin stimulation of glucose transport in primary cultures of rat skeletalmuscle.Diabetes，48：1922-1929，1999.43. Braiman, L., Sheffi-Friedman, L., Bak, A., Tennenbaum, T., and Sampson, S.R. Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of diabetic skeletal muscle 48: 1922-1929, 1999.

44.Bandyopadhyay,G.，Standaert，M.L.，Kikkawa,U.，Ono，Y.，Moscat，J.，and Farese，R.V.Effects of transiently expressed atypical(zeta，lambda)，conventional(alpha，beta)and novel(delta,epsilon)protein kinase C isoforms oninsulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in ratadipocytes：specific interchangeable effects of protein kinases C-zeta and C-lambda.Biochem.J.，337：461-470，1999.44. Bandyopadhyay, G., Standaert, M.L., Kikkawa, U., Ono, Y., Moscat, J., and Farese, R.V. Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel ( delta, epsilon) protein kinase C isoforms oninsulin-stimulated translation of epitope-tagged GLUT4 glucose transporters in ratadipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda. Biochem. J., 337: 461-4970, 19

45.Formisano，P.，Oriente,F.，Miele，C.，Caruso,M.，Auricchio，R.，Vigliotta，G.，Condorelli，G.，and Beguinot，F.In NIH-3T3fibroblasts,insulinreceptor interaction with specific protein kinase C isoforms controls receptorintracellular routing.J.Biol.Chem.，273：13197-13202，1998.45. Formisano, P., Oriente, F., Miele, C., Caruso, M., Auricchio, R., Vigliotta, G., Condorelli, G., and Beguinot, F. In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor intracellular routing. J. Biol. Chem., 273: 13197-13202, 1998.

46.Wang,Q.J.，Bhattacharyya，D.，Garfield，S.，Nacro，K.，Marquez,V.E.，and Blumberg，P.M.Differential localization of protein kinase C delta by phorbolesters and related compounds using a fusion protein with green fluorescent protein.J.Biol.Chem.，274：37233-37239，1999.46. Wang, Q.J., Bhattacharyya, D., Garfield, S., Nacro, K., Marquez, V.E., and Blumberg, P.M. Differential localization of protein kinase C delta by phorbolesters and related compounds using a fusion protein with green fluorescent protein. J. Biol. Chem., 274:37233-37239, 1999.

　　　　　　　　　　　　　　　　　　序列表Sequence listing

 the

<110>  Tennenbaum, Tamar<110> Tennenbaum, Tamar

            Sampson, SanfordSampson, Sanford

            Kuroki, ToshioKuroki, Toshio

            Alt, AddyAlt, Addy

            Shen, ShlomzionShen, Shlomzion

 the

<120>  用于愈合创伤的方法和药物组合物<120> Methods and pharmaceutical compositions for healing wounds

 the

<130>  28181<130> 28181

 the

<160>  16<160> 16

 the

<170>  PatentIn version 3.2<170> PatentIn version 3.2

 the

<210>  1<210> 1

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  1<400> 1

ggaccacaaa uucaucgcgt t                                               21ggaccacaaa uucaucgcgt t 21

 the

 the

<210>  2<210> 2

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  2<400> 2

cgcgaugaau uuguggucct t                                               21cgcgaugaau uugugguct t 21

 the

 the

<210>  3<210> 3

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  3<400> 3

auucaucgcg cgcuucuuct t                                               21auucaucgcg cgcuucuuct t 21

 the

 the

<210>  4<210> 4

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  4<400> 4

gaagaagcgc gcgaugaaut t                                               21gaagaagcgc gcgaugaaut t 21

 the

 the

<210>  5<210> 5

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  5<400> 5

acaaggcuuc cagugccaat t                                               21acaaggcuuc cagugccaat t 21

 the

 the

<210>  6<210> 6

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  6<400> 6

uuggcacugg aagccuugut t                                               21uuggcacugg aagccuugut t 21

 the

 the

<210>  7<210> 7

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  7<400> 7

ucccuaugga uccaaacggt t                                               21ucccuaugga uccaaacggt t 21

 the

 the

<210>  8<210> 8

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  8<400> 8

ccguuuggau ccauagggat t                                               21ccguuuggau ccauagggat t 21

 the

 the

<210>  9<210> 9

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  9<400> 9

acuuauuccu gaucccaagt t                                               21acuuauuccu gaucccaagt t 21

 the

 the

<210>  10<210> 10

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  10<400> 10

cuugggauca ggaauaagut t                                               21cuugggauca ggaauaagut t 21

 the

 the

<210>  11<210> 11

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  11<400> 11

auccgcagug gaaugaguct t                                               21auccgcagug gaaugaguct t 21

 the

 the

<210>  12<210> 12

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  12<400> 12

gacucauucc acugcggaut t                                               21gacucauucc acugcggaut t 21

 the

 the

<210>  13<210> 13

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  13<400> 13

agaccgacga cugucuguat t                                               21agaccgacga cugucuguat t 21

 the

 the

<210>  14<210> 14

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  14<400> 14

uacagacagu cgucggucut t                                               21uacagacagu cgucggucut t 21

 the

 the

<210>  15<210> 15

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  15<400> 15

ggaaccacaa gcaguauuct t                                               21ggaaccacaa gcaguauuct t 21

 the

 the

<210>  16<210> 16

<211>  21<211> 21

<212>  DNA/RNA<212> DNA/RNA

<213>  人工序列<213> Artificial sequence

 the

<220><220>

<223>  SiRNA寡核苷酸<223> SiRNA oligonucleotides

 the

<400>  16<400> 16

gaauacugcu ugugguucct t                                               21gaauacugcu uggguucct t 21

Claims (10)

Translated fromChinese

1.一种用于诱导或加速损伤的皮肤或皮肤创伤的愈合过程的药物组合物，所述药物组合物包括，作为活性成分的胰岛素分泌细胞，以及设计用于所述药物组合物局部施用的药学可接受的载体。1. A pharmaceutical composition for inducing or accelerating the healing process of damaged skin or skin wounds, said pharmaceutical composition comprising, as an active ingredient, insulin-secreting cells, and a drug designed for topical administration of said pharmaceutical composition pharmaceutically acceptable carrier.2.权利要求1的药物组合物，其中所述创伤选自溃疡，糖尿病相关的创伤，烧伤，晒伤，老化皮肤创伤，角膜溃烂创伤，炎性胃肠道疾病创伤，肠炎性疾病创伤，克罗恩氏病创伤，溃疡性结肠炎，痔疮，大疱性表面松解症创伤，皮肤起疱创伤，牛皮癣创伤，动物皮肤创伤，动物糖尿病创伤，视网膜病创伤，口腔创伤(粘膜炎)，阴道粘膜炎创伤，牙龈病创伤，裂伤，手术切口创伤和手术后粘连创伤。2. The pharmaceutical composition of claim 1, wherein the wound is selected from the group consisting of ulcers, diabetes-related wounds, burns, sunburns, aging skin wounds, corneal ulcer wounds, inflammatory gastrointestinal disease wounds, intestinal inflammatory disease wounds, Rohn's disease wounds, ulcerative colitis, hemorrhoids, surface lysis bullosa wounds, skin blistering wounds, psoriatic wounds, animal skin wounds, animal diabetic wounds, retinopathy wounds, oral cavity wounds (mucositis), vaginal Mucositis trauma, gum disease trauma, lacerations, surgical incision trauma and postoperative adhesion trauma.3.权利要求2的药物组合物，其中所述溃疡选自糖尿病性溃疡，褥疮，静脉溃疡，胃溃疡和HIV相关溃疡。3. The pharmaceutical composition of claim 2, wherein said ulcer is selected from the group consisting of diabetic ulcer, decubitus ulcer, venous ulcer, gastric ulcer and HIV-associated ulcer.4.权利要求1的药物组合物，其中所述细胞被转化以产生和分泌胰岛素。4. The pharmaceutical composition of claim 1, wherein said cells are transformed to produce and secrete insulin.5.权利要求1的药物组合物，其中所述细胞被重组PDX1基因转化，从而所述细胞产生和分泌天然胰岛素。5. The pharmaceutical composition of claim 1, wherein said cells are transformed with a recombinant PDX1 gene such that said cells produce and secrete native insulin.6.权利要求1的药物组合物，其中所述细胞被顺式作用元件序列转化，该序列整合到所述细胞的内源胰岛素基因的上游，从而所述细胞产生和分泌天然胰岛素。6. The pharmaceutical composition of claim 1, wherein said cell is transformed with a cis-acting element sequence integrated upstream of said cell's endogenous insulin gene so that said cell produces and secretes native insulin.7.权利要求1的药物组合物，其中所述细胞被重组胰岛素基因转化，从而所述细胞产生和分泌重组胰岛素。7. The pharmaceutical composition of claim 1, wherein said cells are transformed with a recombinant insulin gene such that said cells produce and secrete recombinant insulin.8.权利要求1的药物组合物，其中所述胰岛素分泌细胞能够形成分泌颗粒。8. The pharmaceutical composition of claim 1, wherein said insulin secreting cells are capable of forming secretory granules.9.权利要求1的药物组合物，其中所述胰岛素分泌细胞是内分泌细胞。9. The pharmaceutical composition of claim 1, wherein said insulin secreting cells are endocrine cells.10.权利要求1的药物组合物，其中所述胰岛素分泌细胞是人源的。10. The pharmaceutical composition of claim 1, wherein said insulin-secreting cells are of human origin.

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